Process of expanding T cells

ABSTRACT

The present disclosure relates to a novel process for expanding T cells, such as autologous T cells, cell populations therefrom, pharmaceutical compositions comprising the said cell populations and use of the cells and compositions for treatment, particular the treatment or prophylaxis of virus infection and/or cancer, for example in immune compromised or immune competent human patients.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/364,592, filed on Sep. 24, 2014, now issued as U.S. patent Ser. No.10/351,824 on Jul. 16, 2019, which is a national phase application under35 U.S.C. § 371 that claims priority to International Application No.PCT/GB2012/050896, filed Apr. 23, 2012, which claims priority to U.S.Provisional Patent Application Ser. No. 61/569,577, filed Dec. 12, 2011.The entire contents of these applications are incorporated by referenceherein in their entirety.

INCORPORATED BY REFERENCE

This application incorporates by reference the contents of an 68.9 kbtext file created on May 6, 2019 and named“0043CON_REPLACEMENT_SequenceListing2.txt,” which is the sequencelisting for this application.

TECHNICAL FIELD

The present invention relates to a novel process for expanding T cells,such as autologous T cells, cell populations therefrom, pharmaceuticalcompositions comprising the said cell populations and use of the cellsand compositions for treatment, particular the treatment or prophylaxisof virus infection and/or cancer, for example in immune compromised orimmune competent human patients.

BACKGROUND

While viruses are widely recognized as a cause of infectious disease,certain viruses are also associated with human cancer. The human immunesystem is central to the control of viral infections and alsomalignancies shown to be related to oncogenic viruses. Within thecomplex array of cells, antibodies and immunomodulatory molecules whichconstitute the human immune system, lymphocytes of thymic origin (Tcells) operate in a central role to control viral infections and cancer.Hence, one approach to prevent or treat virus infections and cancer hasbeen to take T-cells from these patients and stimulate and/or expandthem in vitro before transfusing them back into the patient.

In vivo T cell activation and antigen-specific expansion is generallyconsidered to result from a two signal process wherein the first signalis initiated by the ligation of the T cell receptor/CD3 complex with amajor histocompatibility complex class I or class II molecule (MHC ClassI or MHC Class II) presenting a peptide antigen. The MHC Class I or MHCClass II and peptide complex is expressed on the surface of a cell (theantigen presenting cell or APC). The peptide antigen originates from amolecule within the cell which undergoes endogenous processing and maybe, inter olio, (1) a “self” antigen naturally occurring in the body;(2) a tumour antigen which results from a mutation related to cancer or(3) a viral antigen associated with infection or cancer. The recognitionof the antigen by the T cell receptor is considered the first signal andthe second signal arises from co-stimulation which results from theligation of additional surface molecules on the T cell with additionalmolecules on the APC. The up-regulation and ligation of theseco-stimulatory molecules between the T cell and APC may be necessary toeffect or enhance T cell activation since the first signal may not besufficient alone to achieve this. The two signal activation may lead tothe expansion of the T cell so that greater numbers of theantigen-specific T cells will be available to control the pathogen orcancer giving rise to the immune response. The canonical understandingof this two signal activation is based on the same APC providing boththe first signal and the second signal to the responding T cell suchthat the co-stimulation is directly associated with antigen recognition.The in vitro activation and expansion of T cells has traditionally beena long, complex and resource intensive process. A typical process may,for example take 8-12 weeks and often employs live “target” virus and/orviral vectors to achieve antigen presentation by antigen presentingcells (APC). Generally T cell activation/expansion requires staticconditions rather than stirred or physically agitated culture systems.

One prior art method of culturing antigen specific T cells whichrecognize the LMP1 and LMP2 antigens of Epstein Barr Virus (EBV) may besummarized as follows:

Preparatory Steps

In order to achieve the two signal T cell activation and expansionprocess in a controlled manner, it is useful to create anantigen-presenting cell through transfection of B cells taken from thepatient. This is referred to as establishing an autologouslymphoblastoid cell line and is undertaken through infection of the cellwith EBV (EBV-LCL). It takes about 6-8 weeks to develop this cell lineand thus it is one of the first stages that must be started as part of aculture process which relies upon the EBV-LCL for antigen presentation.It is prepared by culturing B cells from the patient with EBV virus inthe presence of cyclosporine A to inhibit EBV-specific T cell outgrowthand elimination of the LCL.

Prior to the expansion step with the EBV-LCLs, the culture systeminvolves the initial activation and expansion of the LMP-1 and LMP-2specific T cells with autologous dendritic cells which been transducedwith a viral vector Ad5f35-LMP1-LMP2 (encoding the EBV proteins LMP1 andLMP2) (days 0 to 8).

day 9 to 12 the cytolytic T lymphocytes (CTLs) are harvested andre-suspended in fresh medium and re-stimulated with EBV-LCLs transducedwith Ad5f35-LMP1-LMP2.

day 13 to 16 cytolytic T lymphocytes are fed with fresh medium andrecombinant human IL-2

followed by weekly re-stimulation using CTL:LCL and twice weeklyaddition of IL-2 for a 4 to 8 week period.

Dendritic cells for use in the process must also be prepared by takingPBMCs from a patient sample and activating them with IL-4 and GM-CSF toprovide adherent PBMCs. These cells are then transduced with a viralvector Ad5f35-LMP1-LMP2 (i.e. encoding the EBV protein LMP1 and LMP2).Finally the dendritic cells are matured by the addition of IL-1β, IL-6,PGE-1 and TNF-α.

Summary of T Cell Expansion Steps

(Also Referred to as Preparation of Cytotoxic T Lymphocytes (CTLs))

Once prepared the transduced dendritic cells are cultured with freshPBMCs from the patient for a period of about 10 days.

The T cells obtained from this step are then cultured with thetransduced EBV-LCLs for a period of about 1 week.

Then the T cells obtained from the latter step are then cultured withtransduced EBV-LCLs in the presence of IL-2 for a further 10 days toprovide an autologous T cell antigen specific product

This process is repeated until sufficient T cells have expanded.

J Immunother Vol 33, Number 3, April 2010 describes a faster and moreefficient way of culturing the cells over a period of 23 days employinga system from Wilson Wolf known as the GRex™ system.

However, this rapid process still employs the traditional viral stimulifor the cells.

There are various problems with the prior art strategy:

(i) the use of live virus such as EBV and viral vectors has thepotential to cause an immunodominant response against the vector whichmay interfere with efficient generation of target virus specific CT L's;

(ii) the use of live virus is an impediment to progression to phase 3trials due to safety concerns;

(iii) the requirement for B cells to manufacture the EBV-LCL, now thatRituxan (which depletes B cells) has become standard therapy for mostlymphoma patients means the technique cannot be employed for manypatients;

(iv) the duration of manufacturing (a minimum of 6 weeks to establishthe EBV-LCL and another 5 to 7 weeks for CTL expansion) is veryinconvenient, impractical and economically challenging;

(v) the complexity of cell manipulation provides many opportunities forerror and contamination of the product, hence, the principles of goodmanufacturing practice (GMP) are difficult to comply with, and

(vi) the autologous antigen presenting cells used to stimulate the Tcell expansion can express antigens other than the target antigen, whichmay reduce the purity of the antigen-specific T cells which are desiredfor the therapeutic T cell product.

Nevertheless skilled persons have been reluctant to move away from theestablished processes because each step was thought necessary togenerate a product with therapeutic characteristics and in particular togenerate T cell populations that are suitable for recognizing cellsinfected by live viruses and cancers expressing viral antigens, in vivo.

The present disclosure provides a method for the rapid and efficientproduction of antigen specific T cells with specificity to a targetantigen.

SUMMARY OF THE INVENTION

The present disclosure provides a process for in vitro expansion ofantigen specific T cells such as autologous antigen specific T cellscomprising the steps:

a) culturing a population of autologous PBMCs in the presence of:

i) dendritic cells which have been pulsed with a peptide/peptide mixrelevant to a target antigen(s) OR a peptide/peptide mix relevant to atarget antigen(s), and

ii) at least one cytokine, and

b) culturing a population of cells obtained from step a) in the presenceof:

i) dendritic cells which have been pulsed with a peptide/peptide mixrelevant to a target antigen(s) OR autologous antigen presenting T cells(T-APC's) cells which have been pulsed with a peptide/peptide mixrelevant to a target antigen(s) and an artificial co-stimulatory factor,andii) optionally a cytokine, and

characterised in that the process does not employ live virus and/orviral vectors or the use of DNA or RNA encoded antigens in the expansionof the relevant T cell population.

In one embodiment there is provided a process for in vitro expansion ofautologous antigen specific T cells comprising the steps:

a) culturing a population of autologous PBMCs in the presence of:

i) dendritic cells which have been pulsed with a peptide mix relevant toa target antigen(s), and

ii) at least one cytokine, and

b) culturing a population of cells obtained from step a) in the presenceof:

i) autologous antigen presenting T cells (T-APC's) cells which have beenpulsed with a peptide mix relevant to a target antigen(s) and anartificial co-stimulatory factor,

ii) a cytokine, and

characterised in that the process does not employ live virus and/orviral vectors or the use of DNA or RNA encoded antigens in the expansionof the relevant T cell population.

In the method of the present disclosure the PBMCs or dendritic cells instep a) and the antigen presenting cells of step b) are generally pulsed(also referred to as loading) with peptides selected to present epitopesfrom the target antigen. These peptides are discussed in more detailbelow.

We have overcome problems of the prior art by:

eliminating the need to generate EBV-LCL's, and therefore avoid the useof live virus, for antigen presentation (this allows the generation ofantigen specific T cells from patients that have previously been B celldepleted, e.g. by Rituxan treatment)

eliminating the need to use viral vector-, or DNA-, or RNA-encodedantigen to achieve antigen expression and presentation in antigenpresenting cells

providing an option to eliminate the use of DCs for antigen presentation

providing a method for T cell activation in which the stimulatory signalis provided by an autologous cell population and the co-stimulatorysignal is provided by a recombinant cell line or an artificialco-stimulatory complex

providing an efficient and robust 2-step culture process to generate atotal of, for example >10e7 CD3 T lymphocytes with suitable antigenspecificity in three weeks or less.

focusing stimulation of the T cell with specificity for clinicallyrelevant virus antigen, such as EBV antigens that are otherwisedominated by antigens that are not expressed in type 2 latency tumors(lymphoma and NPC).

The presently claimed invention has significant advantages for themanufacture of the autologous T cell products and potentially makes thetherapy available to a wider population of patients. It also minimisedthe amount of time, intervention and resource required to produce atherapeutic product, and also advantageously minimises the opportunityfor contamination.

Moreover, the specificity and properties of the therapeutic productobtained are at least equivalent to the product produced by the priorart methods and in a number of aspects may have improved properties.

Autologous cells from certain patients, such as cancer patients aredifferent from cells obtained from healthy individuals because patientcells often are found to be anergic, i.e. incapable of delivering animmune response against antigens associated with the infection orcancer. Immune suppression is often considered to be systemic whereasanergy is usually described on an antigen-specific basis wherein aspecific clone of T cells is no longer able to deliver an immuneresponse against a target antigen. The cancer microenvironment, forexample can create anergy such that T cells which recognize cancerantigens are no longer functional.

Evidence of the immune anergy or suppression is, for example theinability to clear virus infection and/or the presence of virusassociated cancer cells. In healthy individuals these cells are clearedby the immune system (Teague, R. M., B. D. Sather, J. A. Sacks, M. Z.Huang, M. L. Dossett, J. Morimoto, X. Tan, S. E. Sutton, M. P. Cooke, C.Ohlen, and P. D. Greenberg. 2006. Interleukin-15 rescues tolerant CD8+ Tcells for use in adoptive immunotherapy of established tumors. Nat. Med.12:335-341 and Chemnitz, J. M., D. Eggle, J. Driesen, S. Classen, J. L.Riley, S. bey-Pascher, M. Beyer, A. Popov, T. Zander, and J. L.Schultze. 2007. RNA fingerprints provide direct evidence for theinhibitory role of TGF beta and PD-1 on CD4+ T cells in Hodgkinlymphoma. Blood 110:3226-3233).

Additionally, in patients with Nasopharyngeal Carcinoma (NPC), theresults of autologous T cell immunotherapy with antigen-specificcytotoxic T lymphocytes (CTLs) have been relatively ineffective. In onetrial only 1 of 11 patients had a complete response, and this may beexplained by the inability to reactivate LMP-specific T cells from thesepatients. In fact the inventors hypothesise that NPC anergizes T cellswith specificity for the viral tumour antigens.

Thus in some patients the prior art methods were unable to reactivateappropriate antigen specific T cells adequately.

Efficacy of infused T cells depends not only on their ability torecognize the targeted tumor antigens, but also to recognize multipleepitopes within those antigens to prevent tumor escape due to epitopeloss, virus strain variation and T cell driven mutation. Hence there isa need to develop a manufacturing strategy which reproduciblyreactivates and expands CTLs that recognize a broad repertoire ofepitopes from the antigens, such as LMP1-LMP2-, EBNA1 and BARF1—that areexpressed in NPC and in EBV-positive lymphomas.

Prior to the work by the present inventors, who are leaders in thefield, it was not known whether peptides could be used to generate anautologous antigen specific T cell population for prophylaxis andtreatment of viral infections and cancer associated with viruses. Norwas it known that dendritic cells or T-APCs employed in the presentprocess could be rendered useful as antigen presenting cells employingsaid peptides. What is more the T cell responses to the peptides seem tobe relevant in the context of naturally processed peptides which arerecognized by the immune system.

The present invention represents a very significant advancement in thepreparation of (autologous) antigen specific T cell preparation and thisis likely to result in practical benefits for patients and medicalpractitioners.

The factors that are in important in expanded T cell populations of thepresent disclosure are:

the avidity of the T cells for each epitope recognized,

the number of epitopes recognized within each antigen,

the number of antigens recognized,

the fold expansion of T cells and the frequency of T cells with thedesired specificity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of a prior art process forgenerating EVB specific T cells. This method required the production ofautologous dendritic cells (DC) for the first round ofactivation/expansion and Lymphoblastoid Cell Lines (LCL) for thesubsequent rounds of activation/expansion. Both, Dcs and LCLs weretransfected with adenoviral vectors containing the EBV antigens ofchoice in order to stimulate the growth of EBV specific T cells.

FIG. 2 shows a diagrammatic representation of the various embodiments ofthe invention. The diagram shows the process of the invention bydemonstrating the use of peptides to generate EBNA1, LMP1 and LMP2specific T cells. In all new methods the use of Adenoviral vectors asthe way of providing the antigen(s) was replaced by the addition ofexogenous peptide(s). Also the use of LCL for the second round ofactivation/expansion was abolished and replaced with either peptideloaded autologous DCs or peptide loaded antigen presenting autologous Tcells (T-APC) and aK562 cells. Another embodiment shows that the firststimulation can be performed without the use of DC and utilizes theantigen presenting cells present in the PBMC population.

-   -   The generation of PBMCs, DCs and T-APCs is described in this        document under Protocol 1, 2 and 3 respectively.

FIGS. 3A-3E show the stimulation of T cell expansion by aK562 usingT-APCs in step b) of claim 1 of the invention. This demonstrates the useof a novel system to achieve a powerful antigen specific T cellstimulation for the second round of activation/expansion. It is based onthe surprising discovery that the first (antigen specific, stimulatory)signal and the second (co-stimulatory) signal can be provided by twoseparate components. In the example shown the first signal is providedby peptide loaded T-APCs and the second signal is provided by the aK562cells that have been modified to present co-stimulatory molecules butnot MHC. This is as explained in step b) of claim 1 of the invention.

FIG. 3A shows the expansion of T cells using aK562 and T-APCs accordingto the second stimulation in the invention.

FIG. 3B shows the expansion of T cells using aK562 and T-APCs accordingin step b) of claim 1 of the invention. This Figure shows the result in6 normal donors of using the different ratios of CTL:T-APC:aK562 duringthe second stimulation of the process and the resulting cell expansion.The 1:1:5 ratio was shown to be the most optimal in the majority ofdonors.

FIG. 3C shows the optimal CTL to aK562 to T-APC ratio for T cellexpansion in step b) of claim 1 of the invention. The results weregenerated by comparing (C) the fold expansion (as individuallydemonstrated in FIG. 3A), (D) the percentage of CD56+, CD3− cells in theculture and (E) the response in an Interferon-y (IFNg) ELISPOT of thefinal cell product generated using the different cell to cell ratios(SCF=Spot forming colonies).

FIG. 3D shows a comparison of interferon gamma secreting cells incultures of EBV specific T cells employing various ratios of CTLs toT-APCs to aK562 cells and different antigens. The number of cells in thefinal culture producing IFNg in an ELISPOT assay is shown in 2individual donors following the second stimulation at the differentculture ratios. This is shown for the 3 individual EBV antigens ofinterest and a control without antigen.

FIG. 3E shows that T-APC can act as antigen presenting cells for HLAclass I and II restricted antigens.

-   -   A. Upon activation of PBMC with OKT3 and CD28 antibodies        (T-APC), T cells will start to upregulate HLA class II as well        as co-stimulatory molecules such as CD80, CD83, CD86 and 4-1BBL.        Even though HLA class II will reach up to 100% by the end of a        week, the level of co-stimulatory molecules is transient and        remains low.    -   B. aK562 is an HLA(−)ve leukemia cell line that has been        engineered to express stable and high level of CD80, CD83, CD86        and 4-1BBL.

FIGS. 4A-4G show the results using the prior art and the variousembodiments of the invention to expand EBV specific cells from healthydonors

FIG. 4A is a schematic representation of the steps of the prior artprocess and various embodiments of the invention and their nomenclature.This diagram shows the prior art in a shaded box and the nomenclatureused to reference the first and second stimulations. The cell type usedas the APC is shown first and then the way the antigen was delivered (Advector or Peptides—Px). For the embodiment using T-APC and aK562 pluspeptide this is abbreviated in the data to ATC.

FIG. 4B shows expansion of EBV-specific T cells using the prior art andthe various embodiments of the invention. This Figure shows the summaryof results from 9 healthy donors. Following expansion of EBV specific Tcells using the prior art and the 3 other methods outlined in FIG. 4A,cells were counted at day 9, 16 and 23 and the results displayed belowas millions of cells. Both methods employing aK562 in combination withDCs or T-APCs show

FIG. 4C shows a comparison of interferon gamma secreting cells incultures of EBV specific T cells using the prior art and the variousembodiments of the invention. Following culture using the prior artmethod and the various embodiments of the invention, the cellpopulations were re-stimulated with peptides in an ELISPOT assay.Peptides from the three antigens of interest (EBNA-1, LMP1 and LMP2)were used in the assay and the response was either the same or enhancedwhen compared to the prior art. One representative example (of a totalof 9 healthy donors) is shown.

FIG. 4D shows a graph collating the information from all normal donorsfor the embodiments using ATC.

FIG. 4E shows the T cell receptor affinity for EBV specific T cellsexpanded by the prior art and the various embodiments of the invention.Cells were cultured to day 16 using the different methods outlined inFIG. 4A they were then re-stimulated with increasing dilutions ofpeptide in an ELISPOT assay to determine if the new embodiments of theinvention would be detrimental to the avidity of the T cell receptors topeptide. In fact the new methods show a similar and in the case of EBNA1significantly increased avidity for the individual EBV peptides.

FIG. 4F shows the distribution of various T cell markers for cells thatwere expanded using the prior art and the various embodiments of theinvention. At day 16 T cells produced using the 4 different methods wereharvested and immune-phenotyped using flow cytometry. This shows thatthe composition of the cell products is unchanged between the prior artmethod and the embodiments of the invention. There is a differencebetween the prior art and the new embodiments in terms of the expressionof CD62L. This is expressed on naïve and central memory T cells and isdown regulated by effector memory T cells therefore this again could beseen as an advantage for the embodiments of the invention.

FIG. 4G shows that culture of cells using the prior art skews responsestowards antigens expressed in LCL cells rather than on the latency type2 antigens present in EBV+ cancer cells from lymphoma and NPC patients.Cells were generated using the prior art and embodiments of theinvention. These were then re-stimulated with a variety of EBV peptidesin an ELISPOT assay. This shows that the prior art method skews theresponse towards specific LCL dominant epitopes whereas the new methodsshow increased activity against cancer associated antigens such asEBNA1.

FIGS. 5A-5G show the results using the prior art and the variousembodiments of the invention to expand EBV specific cells from NPCpatients

FIG. 5A is a schematic representation of the steps of the prior artprocess and various embodiments of the invention and their nomenclature.This diagram shows the prior art in a shaded box and the nomenclatureused to reference the first and second stimulations. The cell type usedas the APC is shown first and then the way the antigen was delivered (Advector or Peptide—Px). For the embodiment using T-APC and aK562 pluspeptide this is abbreviated in the data to ATC.

FIG. 5B shows expansion of EBV-specific T cells using the prior art andthe various embodiments of the invention. Following expansion of EBVspecific T cells using the prior art and the 3 other methods outlined inFIG. 5A, cells were counted at day 9, 16 and 23 and the resultsdisplayed below as millions of cells or fold expansion. This isrepresentative of 3 and 4 NPC donors respectively. Both methodsemploying aK562 in combination with DCs or T-APCs show significantlybetter expansion than the prior art.

FIG. 5C shows the expansion of EBV-specific T cells by fold.

FIG. 5D shows a comparison of interferon gamma secreting cells incultures of EBV specific T cells using the prior art and the variousembodiments of the invention. Following culture using the prior artmethod and the various embodiments the cell populations werere-stimulated with peptides in an ELISPOT assay. Peptides from the threeantigens of interest were used-EBNA-1, LMP1 and LMP2 and the response inNPC patients was enhanced when compared to the prior art. Onerepresentative example is shown.

FIG. 5E shows the T cell receptor affinity for EBV specific T cellsexpanded by the prior art and the various embodiments of the invention.Cells were cultured to day 16 using the different methods outlined inFIG. 5A. They were then re-stimulated with increasing dilutions ofpeptide in an ELISPOT assay to determine if the new embodiments of theinvention would be detrimental to the avidity of the T cell receptors topeptide. In fact the new methods show a similar and in the case of EBNA1significantly increased avidity for the individual EBV peptides.

FIG. 5F shows the distribution of various T cell markers for cells thatwere expanded using the prior art and the various embodiments of theinvention. At day 31T cells produced using the 4 different methodsdescribed in FIG. 5A were harvested and immune-phenotyped using flowcytometry. This shows that the composition of the cell products inunchanged between the prior art method and the embodiments of theinvention. There is a difference between the prior art and the newembodiments in terms of the expression of CD62L. This is expressed onnaïve and central memory T cells and is down regulated by effectormemory T cells therefore this again could be seen as an advantage forthe embodiments of the invention.

There is some change as to the methods outlined in FIG. 5A. This is dueto the NPC patient cells being subjected to more than one re-stimulationstep. However the nomenclature for each stimulation remains the same.This data is representative of 4 NPC patients.

FIG. 5G shows that T Cells from NPC patients expanded employing the newembodiments of the invention (CD3+/CD19-) kill LCL (EBV+ cancercell-line, CD3-/CD19+) better in co-culture for 4 and 10 days as T cellsexpanded by the prior art. T cells and LCL were incubated at a 1:1ratio.

FIGS. 6A-6E show the results using the prior art and the variousembodiments of the invention to expand EBV specific cells from lymphomapatients

FIG. 6A is a schematic representation of the steps of the prior artprocess and various embodiments of the invention and their nomenclature.This diagram shows the prior art in a shaded box and the nomenclatureused to reference the first and second stimulations. The cell type usedas the APC is shown first and then the way the antigen was delivered (Advector or Peptides—Px). For the embodiment using T-APC and aK562 pluspeptide this is abbreviated in the data to ATC as previously but alsohas been alternatively abbreviated to KATpx and ATpk in this section ofthe data.

FIG. 6B shows expansion of EBV-specific T cells using the prior art andthe various embodiments of the invention. Following expansion of EBVspecific T cells using the prior art and the 3 other methods outlined inFIG. 6A, cells were counted at day 9 and 16 and the results displayedbelow as millions of cells or fold expansion. This is representative ofresults with cells from 4 lymphoma patients.

FIG. 6C shows expansion of EBV-specific T cells by fold.

FIG. 6D shows a comparison of interferon gamma secreting cells incultures of EBV specific T cells using the prior art and the variousembodiments of the invention. Following culture using the prior artmethod and the various embodiments the cell populations werere-stimulated with peptides in an ELISPOT assay. Peptides from the threeantigens of interest were used—EBNA-1, LMP1 and LMP2 and the response inlymphoma patients was enhanced when compared to the prior art. Onerepresentative example is shown. These results show that when peptidepulsed PBMCs or DCs were used for the first expansion, the number ofantigen specific T cells was significantly increased after 9 daysrelative to culture where the prior art was used.

FIG. 6E shows that T Cells from lymphoma patients expanded employing thenew embodiments of the invention (CD3+/CD19−) kill LCL (EBV+ cancercell-line, CD3−/CD19+) equally well or better in co-culture for 2 or 4days as T cells expanded by the prior art. T cells and LCL wereincubated at a 1:1 ratio.

FIGS. 7A-7C show the results using the prior art and the variousembodiments of the invention to expand VZV specific cells from healthydonors. PBMCs were pulsed with overlapping peptide libraries (15 mersoverlapping by 11 amino acids) (pepmixes) spanning the VZV proteins, gE,ORF10. 1E61, 1E62 and 1E63 in the presence of IL-4 and IL-7. On days 9,16 and 23 they were restimulated with autologous activated T cells(AATCs, T-APCs) pulsed with the same pepmixes in the presence of aK562cells expressing co-stimulatory molecules CD80, CD83, CD86 and 4-1BBligand (K562-cs), at a ratio of CTLs to T-APCs to K562-cs of 1:1:5.Their rate of proliferation was measured by counting andantigen-specificity was measured in gamma interferon ELISPOT assays.

FIG. 7A is a schematic representation of the steps of the prior artprocess and various embodiments of the invention and their nomenclature

FIG. 7B shows expansion of EBV-specific T cells using the prior art andthe various embodiments of the invention. Growth rate of T cellsexpanded using the protocol described above. >500 fold expansion can beachieved over 22 days

FIG. 7C shows a comparison of interferon gamma secreting cells incultures of EBV specific T cells using the prior art and the variousembodiments of the invention. Frequency of T cells from 6 to 9 healthyVZV-seropositive donors that secrete gamma interferon in response tostimulation with VZV peptides after activation with pepmix pulsed PBMCsor DCs on day 0 and pepmix-pulsed autologous activated T cells (T-APCs)plus K-562-cs cells on days 9, 16 and 23. Each symbol represents onedonor. Embodiments of the current invention either using peptide pulsedPBMCs or DCs during the first expansion and T-APCs in combination withaK562s for subsequent expansions result in a significant expansion oftarget CTLs across the various VZV antigens used.

DETAILED DESCRIPTION OF THE INVENTION

Autologous T cells are cells derived from the patient i.e. cells thatare natural to the patient as opposed to cells from a donor. Certaintumours associated with viral infection have developed a way to grow inthe patient despite the presence of virus-specific T cells. Thisinvolves the expression of molecules that are inhibitory to T cells andthe modulation of virus gene expression. Thus the T cells in thesepatients may have reduced function towards the cancer cells, which maybe described as a form of anergy. In autologous T cell therapy a sampleof T cells are removed from the patient for activation and expansion exvivo. Once the antigen-specific T cell population has been prepared itis infused into the patient where the T cell cells will further expandand will eliminate cells presenting their target antigens by direct(cytotoxic) and indirect (immune regulatory) mechanisms.

“T cell” is a term commonly employed in the art and intended to includethymocytes, immature T lymphocytes, mature T lymphocytes, resting Tlymphocytes or activated T lymphocytes. A T cell can be a T helper (Th)cell, for example a T helper 1 (Th1) or a T helper 2 (Th2) cell. The Tcell can be a CD4+ T cell, CD8+ T cell, CD4+CD8+ T cell, CD4−CD8− T cellor any other subset of T cells.

Antigen specific T cell as employed herein is intended to refer to Tcells that recognise a particular antigen and responds thereto, forexample by proliferating and/or producing cytokines in response thereto.

In one or more embodiments the process does not employ recombinanttarget antigens for stimulating specificity. Recombinant antigens hereinrefers to whole or large fragments of antigens prepared by recombinanttechniques. In contrast the peptides employed are small fragments ofantigen and are generally synthetic.

The present invention relates to ex vivo processing of cells and the Tcell products obtained therefrom. Usually the present invention does notinclude the step of obtaining the sample from the patient. The step ofobtaining a sample from the patient is a routine technique of taking ablood sample (which can be processed and optionally provided as anapheresis product). This process presents little risk to patients anddoes not need to be performed by a doctor but can be performed byappropriately trained support staff. In one embodiment the samplederived from the patient is approximately 200 ml of blood or less, forexample 150 ml or less such as in the range 100-150 ml. Generally atleast about 60 ml of blood is required.

Typically the PBMCs for T cell expansion, DC generation and T-APCgeneration are obtained from the blood or apheresis product by Ficolldensity gradient separation known to those skilled in the art. Ficolldensity gradient separation employs a synthetic sucrose polymer theconcentration of which varies through the solution to exploit theseparation of different cells during sedimentation. Suitable reagentsare available, for example from GE Healthcare, such as Ficoll Paque™PLUS.

In one embodiment the centre responsible for taking the blood sample orfor shipping the blood sample processes the sample by Ficoll densitygradient separation prior to transportation.

In one embodiment the blood sample or processed blood sample istransported at ambient temperature, for example above 4° C. and belowabout 30° C.

In one embodiment the blood sample or processed blood sample is filledinto a container, such as bag, comprising two chambers, wherein onechamber contains additives, such as preservatives and/or anticoagulantsand the blood or processed blood is filled into the second chamber,after which a seal between the first and second chamber is broken andthe contents of the two chambers are mixed. Culturing cells as employedherein is intended to refer to activating and expanding and/ordifferentiating cells in vitro.

It is known to the skilled person, that expansion of T cells isgenerally performed in a suitable T cell expansion medium. Generally theprocess of step a) can be performed without changing the medium.Generally the process of step b) can be performed without changing themedia. However, media should be changed if indicated by a glucometer,for example that is if the glucose in the system falls below 100 mg/dl.Thus the process of the present disclosure is efficient in that itminimizes the amount of intervention required to expand the T cells.

T cell expansion may be evaluated by counting viable CD3+ cells.

Viable cells can be tested by cell staining with, for example Trypanblue (and light microscopy) or 7-amino-actinomycin D, vital dye emittingat 670 nm (or Via Probe a commercial ready-to-use solution of 7AAD) andflow cytometry, employing a technique known to those skilled in the art.Where the stain penetrates into the cells the cells are considered notviable. Cells which do not take up dye are considered viable. Anexemplary method may employ about 5 μL of 7AAD and about 5 μL ofAnnexin-V (a phospholipid-binding protein which binds to externalphospholipid phosphatidylserine exposed during apotosis) per approximate100 μL of cells suspension. This mixture may be incubated at ambienttemperature for about 15 minutes in the absence of light. The analysismay then be performed employing flow cytometry. See for example M GWing, A M P Montgomery, S. Songsivilai and J V Watson. An ImprovedMethod for the Detection of Cell Surface Antigens in Samples of LowViability using Flow Cytometry. J Immunol Methods 126: 21-27 1990.

T cell expansion media generally comprises serum, media and anycytokines employed in the relevant expansion step (i.e. step a) or stepb)).

In one embodiment the serum employed is, for example 15% serum or lesssuch as 10% serum, in particular human serum is employed.

In one embodiment the media is Advanced RPMI or RPMI 1640, availableform Life Technologies.

In one embodiment the cytokines employed are discussed below.

In one embodiment the cell expansion medium comprises 10% human ABserum, 200 mM

L-glutamine, 45% Earle's Ham's amino acids (EHAA or Click's medium) and45% advanced RPMI or RPMI-1640.

In one embodiment the media employed does not require the use of serum.

Cell expansion as employed herein refers to increasing the number of thetarget cells in a population of cells as a result of cell division.

In one embodiment in step a) the PBMCs are treated directly with apeptide/peptide mix. It was very surprising that autologous PBMCs couldactivate T cells in the presence of peptides in a manner similar to whenautologous dendritic cells are present, in particular because themessage from the literature is that dendritic cells are the optimalantigen presenting cells. Chen M L, Wang F H, Lee P K, Lin C M. ImmunolLett. 2001 Jan. 1; 75(2):91-6.

In another embodiment in step a) of the present process dendritic cellsare employed which are prepared from the patients PBMCs.

The blood sample is not generally subject to initial physical selectionof cells, for example selection of a sub-population of cells (or Tcells) from an apheresis population.

In one embodiment 1 to 2×10⁷ PBMCs are stimulated with 0.5 to 1×10⁶peptide-pulsed DCs in the presence of cytokines in the GRex40 in 30 mlsof medium. A harvest of cells, for example in the range 50 to 80×10⁷antigen-specific responder T cells after 9 to 14 days of culture.However this may be lower in patients with anergic T cells.

Medium (45% advanced RPMI, 45% EHAA, 10% FCs and 200 mM L-glutamine)will be changed only if indicated by a drop in glucose below 100 mg/dl(on glucometer).

Dendritic cells are often referred to, by those skilled in the art, asprofessional antigen presenting cells. The term refers to the fact thatdendritic cells are optimal in delivery the two signal activationprocess to T cells, i.e., in addition to presenting antigen on the cellsurface, dendritic cells also provide a strong co-stimulatory signal.Both signals, stimulation by antigen presentation and co-stimulation arerequired to achieve T cell activation.

Dendritic cells for use in the process of the present invention may begenerated from a sample of the patients PBMCs by pulsing (or loading)with a peptide mixture the details of which are discussed below. Pulsingor loading as employed herein is intended to refer simply to exposingthe relevant cells, such as PBMCs or dendritic cells, to the peptide mixin an appropriate medium.

Dendritic cells for use in the process may be prepared by taking PBMCsfrom a patient sample and adhering them to plastic. Generally themonocyte population sticks and all other cells can be washed off. Theadherent population is then differentiated with IL-4 and GM-CSF toproduce monocyte derived dendritic cells. These cells may be matured bythe addition of IL-1β, IL-6, PGE-1 and TNF-α (which upregulates theimportant co-stimulatory molecules on the surface of the dendritic cell)and are then transduced with a peptide mixture as described herein toprovide the required dendritic cells. Reference to generating andmaturing DC in this way is found in Jonuleit H, Kuhn U, Muller G, et al.Pro-inflammatory cytokines and prostaglandins induce maturation ofpotent immunostimulatory dendritic cells under fetal calf serum-freeconditions. Eur J Immunol. 1997; 27:3135-3142.

Peptides may be added at 1 to 100 ng peptide/15×10⁶ PBMCs or ATCs (seediscussion below) or 1 to 100 ng peptide per 2×10⁶ DCs for each peptidelibrary/pepmix.

In one embodiment PBMCs are stimulation with IL-4 and GM-CSF for 3 to 5days followed by 1 or 2 days of maturation with GM-CSF, IL-4, TNF-a,IL-Ib, PGE-1 or PGE-2 and IL-6 followed by pulsing with said peptides.

Thus in one embodiment the dendritic cells in step a) are autologous.

In one embodiment the dendritic cell response produced is balanced, CD4and CD8 response.

Balanced CD4 and CD8 response as employed herein is intended to refer tothe fact that the CD4 cells or CD8 are not depleted in the expansionprocess. However a balanced population as employed herein may still beskewed in that there may be more CD4 cells than CD8 cells or vice versa.

In one embodiment the ratio of PBMCs to dendritic cells in step a) inthe range 10:1 to 50:1 respectively, for example 15:1, 16:1, 17:1, 18:1,19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, such as about 20:1.

Dendritic cells provide powerful activation of T cells and when thefrequency of antigen-specific T cells in the culture is low (less than 1in 100 T cells may be specific for the antigens of interest), forexample in step a) it is efficient to employ dendritic cells. However,as the numbers and frequency of antigen specific T cells expand and theratio of specific T cells to dendritic cells increases the efficiency ofthe activation of the dendritic cells decreases unless the number ofdendritic cells can be increased. The antigen-specific T cells willfrequently deliver a cytotoxic response to the dendritic cell followingactivation. The killing of the dendritic cells will then reduce theactivation signal available to continue activating and expanding thetarget antigen-specific T cells.

Whilst dendritic cells are very effective in stimulating T cells toexpand into populations specific to a target antigen it is difficult togenerate large quantities of dendritic cells. Thus whilst step b) mayemploy dendritic cells in practice there are advantages to employingdifferent antigen presenting cells and an artificial co-stimulatorfactor in step b). In some instances when the number of dendritic cellsto T cells is too small no activation of the T cells is observed. Thisis discussed in more detail below. Advantageously, dendritic cells arethought to be capable of activating both memory T cells and naïve Tcells. The presence of memory T cells in the cell populations accordingto the present invention may be important.

Minor population as employed herein is intended to refer to the factthat the absolute numbers of cells in the minor population issignificantly lower than the number of cells in the desired population,for example 30 percent or less of the total population.

The peptide mixtures described below may be used for one or morepurposes selected from pulsing of dendritic cells, pulsing of antigenpresenting cells or may be employed directly with PBMCs in step a). Thepeptide mixes are from a relevant viral antigen, for example an EBVviral antigen. Epstein-Barr virus, frequently referred to as EBV, is amember of the herpes virus family and chronically infects over 95% ofthe world population.

In one embodiment the peptides are from an antigen of papilloma virus.

In one embodiment the peptides are from an antigen of hepatitis C virus.

In one embodiment the peptides are from an antigen of vaccinia virus(VV).

In one embodiment the peptides are from an antigen of varicella zostervirus (VZV).

In one embodiment the peptides are from an antigen of humanimmunodeficiency virus.

In one embodiment the peptides are from an antigen of Hepatitis B,HHV-8, CMV, HTLV-1, SV40 and/or merckel cell virus.

In one embodiment the peptides are from a combination of viruses, forexample any two or more described herein, such as EBV and VZV, EBV andVV, VZV and VV or EBV, VZV and VV.

Instead of culturing the autologous T cells in the presence of cellsthat are infected with the relevant virus, such as EBV, or in thepresence of adenovirus vectors encoding viral proteins the cells arecultured in the presence of antigen presenting cells that were pulsedwith a peptide or a mixture of peptides. This reduces the risk ofcontamination of the final product with pathogens which is importantbecause there is no method of sterilizing the T cell product that willbe infused into the patient. In one embodiment the peptide mix or someof the peptides in the mix cover part or all of the sequence of theantigen LMP1 (Latent Membrane Protein 1 Uniprot number P03230).

In one embodiment the peptide mix or some of the peptides in the mixcover part or all of the sequence of the antigen LMP2 (Latent MembraneProtein 2 Uniprot number Q1HV.12).

In one embodiment the peptide mix or some of the peptides in the mixcover part or all of the sequence of the antigen EBNA 1, 2, 3, 4, 5 or 6or a combination of the same, in particular EBNA 1.

Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional, dimericviral protein associated with Epstein-Barr virus. It is the only viralprotein of EBV that is found in all EBV-related malignancies andtherefore is a significant antigen to target. It is important inestablishing and maintaining the altered state that cells take wheninfected with EBV. EBNA1 possesses a glycine-alanine repeat sequencethat separates the protein into amino- and carboxy-terminal domains.This sequence also seems to stabilize the protein, preventingproteasomal breakdown, as well as impairing antigen processing and MHCclass I-restricted antigen presentation. This thereby inhibits theCD8-restricted cytotoxic T cell response against virus-infected cells.The EBNA1 transcript area originates at the Qp promoter during latencyphases I and II. It is the only viral protein expressed during the firstlatency phase. The EBNA1 pepmix activates HLA class II-restrictedcytotoxic CD4 T cells.

In one embodiment the peptide mix or some of the peptides in the mixcover part or all of the sequence of the antigen BARF 1 (BamHI Arightward reading frame 1, Uniprot number Q777A5). BARF1 is a 221 aminoacid protein encoded by the BARF 1 gene which is located in the BamHI-Afragment of the EBV genome. BARF1 is expressed in various EBV-associatedepitheloid malignancies, for example in NK/T lymphomas and in Burkitt'slymphoma.

Other potential EBV antigens include LP and BHRF1.

The major virion/envelope proteins for vaccinia virus are described inPNAS January 7, 203 vol 100 no. 1 page 217-222 (Drexler et al). Theseinclude A1OL (major core protein p4a), H3L (heparin binding protein),C7L (host range protein 2), D8L (cell surface binding protein), B22R(unknown function) and G5R (unknown function).

Varicella zoster virus immunogens include gE, ORF10, 1E61, 1E62 and1E63.

Peptides from each one of the specific target antigens listed supra mayindependently be employed in step a) and/or step b) of the process.

Generally some or all of the epitopes/antigens/peptides employed orexpressed for the purpose of providing a primary signal for T activationin step a) and step b) will be common to both steps. The peptides maycover part or all of the target antigen, for example may be overlappingto increase the opportunity of presenting the amino acids of an epitopein an immunologically relevant way. Alternatively or additionallypeptides of known epitopes may be included and if desiredover-represented, that is to say may be a more significant percentage ofthe peptides presented.

Antigens in HIV include gag, pol, env, nef, gp180, gp120 and the like.

“Covers part or all of the sequence of the antigen” as employed hereinis intended to refer to the fact that there is identity or significantsimilarity between the peptide and the relevant portion of the fulllength antigen, for example the peptide is substantially identical to acontiguous region of the antigen.

Selected to present epitopes as employed herein is intended to refer tothe fact that the linear sequence of an epitope is known and includedinto a peptide mix (that is peptides are selected encoding knownepitopes) or, for example where the antigen sequence has not beenepitope mapped then the peptides are designed to cover part or all ofthe sequence in an overlapping manner to maximise the opportunity ofpresenting one or more appropriate epitopes. Of course a mixture ofthese two strategies can be employed if desired, for example knownepitopes may be represented to a greater extent in a peptide mixture.

In one embodiment the peptides in the mix are from one or more relevantviral antigens, for example one, two, three, four or more.

In one embodiment the peptide mix comprises or consists of sequencesfrom at least LMP1 and LMP2. In addition in one embodiment EBNA1 and/orBARF1 are added to the antigen mixture to reduce the chances of immuneescape by mutation or down-regulation of viral antigens.

Peptide as employed herein intended to refer to short polymers of aminoacids linked by peptide bonds, wherein the peptides contain at least 2but generally not more than 50 amino acids.

The peptides employed are sufficiently long to present one or morelinear epitopes, for example are on average 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 amino acids long.

In one embodiment some of the peptides of the mixture overlap (inrelation to the sequence of a single antigen), that is to say that theyare from a single antigen and are arranged such that portions of thefragments and certain sequence of amino acids from the parent sequenceoccur in more than one peptide fragment of the mix. The overlap of thepeptides means that there is redundancy in the amino acid sequence.However, this method maximises the opportunity to present epitopes fromthe parent antigen in an appropriate manner, particularly when epitopemapping information is not available for the parent antigen.

In one embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15amino acids overlap in each peptide. In one embodiment the peptides inthe libraries for each protein are 15 amino acids long and overlap by 11amino acids so that all potential HLA class I epitopes can be presentedfrom a protein. The peptides can be longer, for example 20 amino acidsoverlapping by 15 or 30 amino acids overlapping by 25.

Examples of suitable peptides sequences include in the sequence listingfiled herewith.

In one embodiment the peptide mix comprises or consists of 2-1000peptides, more specifically 2-500, for example 2-400, 2-300, 2-200 or2-100 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more peptides.

The peptides of step a) or the peptides employed to create antigenspecific dendritic cells in step a) or b) or employed to prepare theantigen presenting cells of step b) of the process are independently,selected based on the criteria above. However, the process is mostefficient where the some or all of the peptides employed or thematerials such as dendritic cells and/or antigen presenting cellsemployed are pulsed with some or all of the same epitopes. In thissituation step b) then reinforces and augments the responses generatedin step a).

The peptide mixes described above may also be used to generate antigenpresenting T cells (T-APCs) which are employed in some embodiments instep b) of the process. To prepare the antigen presenting T cells, theyare generated form PBMCs as described below and are pulsed with arelevant peptide mix, for example peptides are added at 1 to 100 ngpeptide/15×10⁶T-APCs for each peptide library. Each library as employedherein may refer to the peptides made for each antigen.

In one embodiment the antigen presenting cells are autologous.

Activated T cells express HLA class I and upregulate class II antigensas well as CD80 and CD86 (transiently).

In one embodiment after pulsing with peptides to provide the specificT-APCs the latter are irradiated to ensure that they don't expand anyfurther when they are employed in step b).

Irradiation may be effected employing a source of gamma radiation or asource of X-rays.

In one embodiment on day 9 to 14, about 5×10⁷ responder T cells fromstep a) are stimulated with 5×10⁷ irradiated T-APCs and 5×10⁷ aK562s ina GRex 500 in 400 mls of medium containing IL-15 for up to 14 days.

US 2003/0147869 discloses that the aK562 cells described therein may beengineered to render them antigen presenting cells. These cells intheory could be expressed in significant numbers. One may think thatthese could be employed as an alternative to T-APCs of step b). Howeverthese aK562 antigen presenting cells do not express HLA and if they didthe HLA phenotype would not match the effector T cell restrictionpattern and they would activate alloantigen-specific T cells. Thepresent inventors have found that in the absence of HLA-expressing cellsthere is poor activation of the relevant target T cell population.Whilst in theory these aK562 cells could be engineered to express HLAthis would be need to be matched to the patient in each case therebymaking the process unnecessarily complicated and expensive.

There are 7,196 HLA alleles. These can be divided into 6 HLA class I and6 HLA class II alleles for each individual (on 2 chromosomes). They canbe mixed and matched in any way and therefore introducing theappropriate combination of HLA alleles into aK562 cells to (1)reactivate all potential T cells and (b) induce no allo-reactivity wouldbe impossible at this moment in time.

The T-APCs according to the present invention present on average atleast one epitope from a target antigen and for example may present 2,3, 4, 5, 6 or more epitopes.

In one embodiment the T-APCs present epitopes from more than one targetantigen, for example 2, 3, 4 or more target antigens.

In one embodiment the ratio of cells (or CTLs) obtained from step a) toT-APCs is in the range 4:1 to 1:2, for example 1:1. A high proportion ofT-APCs maximises the efficiency of the expansion. Usually it isdifficult to generate dendritic cells is such high proportions, whichmeans the time taken for expansion of the relevant T cell population maybe longer with dendritic cells than the time taken for expansion withantigen presenting cells. In some instances where the numbers of thedendritic cells are very low the expansion may not occur at all. Thusthe process employing antigen presenting cells in step b) may beadvantageous in that the periods taken for expansion are shorter andthus provide a more efficient process.

Thus in one embodiment T-APCs are employed in step b) of the presentmethod.

The use of T-APCs in the present process replaces the use LCLs in theprior art process. LCL cell lines are immortalised by infection withlive EBV virus. Avoiding the use of LCLs in the present process is ahuge advantage.

LCLs engineered to present other viral antigens through infection withadenovirus vectors, or pulsed with peptides can provide an effectivesecond stimulation, but for weak antigens, both adenoviral and EBVproteins from the vector and the LCL respectively produce strongcompetition, so that the major component of the final CTL product isoften specific for adenovirus or dominant EBV antigens expressed in LCLsbut not expressed in type 2 latency tumors (lymphoma and NPC).

The use of simple peptides in step b) does not seem viable because theinventors' experience is that presenting peptide mixes to the CTLsresulting from step a) simply “confuses” the cells and results in thempresenting peptides on their surface. This then results in the CTLsbeing targets for each other and they start to destroy themselves. Thiscauses depletion of the cells which is clearly undesirable and contraryto the purpose of performing the process.

In one embodiment step b) is performed more than once, for example 2, 3,4, 5 or more times until sufficient numbers of the relevant antigenspecific T cell population are obtained.

Sufficient numbers may, for example be sufficient cells to continueexpanding in vivo and stimulating the patient's immune response to thetarget virus infection and/or target associated cancer, such as 1 to90×10³, 1 to 90×10⁴, 1 to 90×10⁵, 1 to 90×10⁶, 1 to 90×10⁷, 1 to 90×10⁸or more cells, such as 80×10⁷ cells.

In one embodiment there is provided a process wherein if cells do notexpand sufficiently after step b) they may receive additionalstimulation with:

(1) peptide-pulsed, irradiated autologous activated T cells andirradiated co-stimulatory aK562 cells,

(2) irradiated PBMCs from blood bank approved allogeneic donors and/or

(3) submitogenic doses of anti-CD3; 1 to 100 ng per mL for example 50 ngper ml.

T-APCs are not professional antigen presenting cells. Therefore, inaddition to the antigen presentation provided by the T-APCs aco-stimulator factor is required to stimulate T cell expansion anddifferentiation.

An artificial co-stimulatory factor as employed herein is an exogenousfactor which is added to the culture to provide a T cell activationsignal to complement the T-APC antigen presentation signal, for examplewhere together the co-stimulatory factor and the T-APC antigenpresentation signal stimulates or facilitates the autologous T cellsexpanding in a specific manner i.e. that stimulates the targetpopulation of cells as they expand to be specific for the targetantigen, wherein the specificity aspect is elicited by the T-APC. Anexogenous factor is one that is not present in the culture of PBMCswithout addition or where the naturally occurring amounts present in thecell culture are low and are augmented by addition of exogenous amountsof the factor.

Beads bearing CD80/86 may be employed as a co-factor. Beads withanti-CD28 (ligand for CD80/86) and anti-4-1BB are available but theyalso contain anti-CD3 which eliminates the desired antigen mediatedspecificity of the expansion. Thus beads with anti-CD28 (ligand forCD80/86) and anti-4-1BB in the absence of anti-CD3 form an aspect of thepresent disclosure.

In one embodiment the artificial co-stimulatory factor is a cell orcell-line engineered to express particular protein(s) on its surface orassociated with its surface (see US 2003/0147869 for association ofcertain antibodies on the surface of the cell), for example a HLAnegative cell line and which has been genetically modified to expressco-stimulatory molecules, such as the aK562 cell-line as disclosed inU.S. Pat. No. 7,745,140. Cell lines such as the latter may be employedin the process of the invention to provide a prolonged co-stimulatorysignal.

Thus in one embodiment the cell line such as aK562 does not express HLA.

Thus in one embodiment the cell line such as aK562 does not expressantigen.

aK562 cell as employed herein may refer to the original cell linedescribed in U.S. Pat. No. 7,745,140. However, for the purposes of thepresent process generally an anti-CD3 antibody will not usually beloaded onto the FcY receptor on the surface thereof. Preferably theaK562 cell as referred to herein is a derivative of the original aK562cell line comprising at least one, such as one, two, three or fourco-stimulatory factors on the surface thereof, in particularindependently selected from the group comprising an anti-CD28 antibody,an anti-CD80 antibody, an anti-CD86 antibody and 4-1BBL.

One or more co-factors may have a role to play in reducing apoptosis ofT cells and inducing a proliferative cycle of, for example about 7 to 10cell divisions.

The cell is engineered to express co-stimulatory factors on its surfacethat provide signals that are important in the stimulation (activationor differentiation) or survival of T cells and complement the signalgenerated by the presentation of antigens on the surface of antigenpresenting cells. Examples of the molecules that may be expressed on thecell line surface selected from the group comprising CD80, CD86, CD83,4-1BB-ligand, and a combination thereof, for example 1, 2, 3, or 4thereof. The four elements together provide a powerful co-stimulatorysignal.

In one embodiment the aK562 cell further expresses OX-40 ligand on itssurface.

These cells act together with the T-APCs, described above, to provideall the signals necessary for T cell activation.

CD80 and CD86 bind CD28, a surface glycoprotein present on about 80% ofperipheral T cells in humans. In combination with the activation of theT cell receptor, this binding provides potent stimulation of T cells.What is more CD28 (on T cells) binding to its ligand in conjunction withT cell receptor engagement induces the production of IL-2.

In one embodiment the culture in step b) comprises only endogenous IL-2.

Alternatively the artificial co-stimulatory factory may be agonisticantibody for a relevant receptor such as antibodies that target forexample CD28. These antibodies may be provided directly into the media,or attached to beads, or may be attached to the surface of the cell line(such as aK562). This is described in detail in US 2003/0147869,incorporated herein by reference.

Providing the co-factors on beads does not lead to the most efficientantigen specific expansion possible. Thus in one embodiment theco-factors are expressed on or associated with a cell such as aK562cell.

This artificial co-stimulatory cell employed in step b) of the presentinvention (such as the aK562) cell seems to be acting by a new andsurprising mechanism. The process of the present disclosure is evidencefor the first time that antigen presentation can be provided on one celland the co-stimulation can be provided by a different cell to stimulateand activate T cells. In the context of generating antigen specific Tcells this is very surprising because in practice in the past generallyaK562 cells have been engineered to express HLA molecules or an antibodythat targets the T cell receptor and model the in vivo systems where theT cell receptor mediated signal and the co-stimulatory signal isprovided by one cell (i.e. the contact is between two cells and forexample CD28 ligation on T cells in conjunction with TCR engagementinduces the production of IL-2 which triggers continued proliferation;June et al 1994, Jenkins et al 1993 and Schwartz 1992). Thus certainaK562 cell lines have been established which express MHC class 1 A2 andA3Britten, C. M.; Meyer, R. G.; Kreer, T.; Drexler, I.; Wolfel, T.;Herr, W. (2002), “The use of HLA-A*0201-transfected aK562 as standardantigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOTassays”, Journal of Immunological Methods 259 (1-2): 95-110, and Clark,R. E.; Dodi, I. A.; Hill, S. C.; Lill, J. R.; Aubert, G.; Macintyre, A.R.; Rojas, J.; Bourdon, A. et al. (2001), “Direct evidence that leukemiccells present HLA-associated immunogenic peptides derived from theBCR-ABL b3a2 fusion protein”, Blood 98 (10): 2887-93. Thus in certainembodiments the co-stimulatory cell such the aK562 cell is effectivelyan autonomous co-stimulatory factor (Third Party Co-stimulatory factor)which together with the T-APCs stimulates the antigen specific expansionof the target T cell population. Whilst in theory a aK562 cell thatexpresses the Fc receptor and that can be loaded with anti-CD3 antibody(such OKT3 antibody), to provide an antigen independent activationsignal, may be employed in the present processes in at least oneembodiment the aK562 (or engineered cell) is not loaded with an anti-CD3antibody because generally the non-specific signal is not desirable. Thepresent disclosure also extends to the use of an engineered cells line,such as aK562 cell, for providing an artificial co-stimulatory signalindependent of antigen presentation in the expansion of T cells, such asautologous T cells.

Thus in one embodiment the process is characterised further in that theprocess of expanding the population of cells in step b) is surprising inthe context of relying upon only (1) T-APCs (2) peptides to pulse/loadthe latter and (3) aK562 cells transduced to express only co-stimulatorymolecules without antigen-specific presentation qualities. This processas described achieves expansion on an antigen-specific basis (asmeasured by an increasing percentage of antigen-specific T cells duringthe expansion process) despite the fact that the aK562 cells have notbeen engineered to present specific antigens. Without the addition ofother APCs in this step (e.g. DCs), the expansion process is thereforerelying upon T-APCs to achieve the first signal activation with respectto presentation of the target antigen. This is unusual since T-APCs areconsidered sub-optimal with respect to both antigen presentation andco-stimulation. The role of aK562 cells to provide co-stimulation and toenhance expansion has been described before, but the previousdemonstration showed general CD3+ T cell expansion at the cost ofreduced antigen specificity since the aK562 cell were not engineered topresent specific antigens. In the present invention, theantigen-specificity is increased with expansion which indicates that thefirst signal antigen recognition step is being achieved by the T-APCwhich is acting synergistically with the second signal co-stimulationfrom the aK562 cell. This bifurcation of the antigen presentation andco-stimulation signals contravenes the currently embraced paradigm forantigen-specific T cell expansion wherein the first signal and secondsignal are delivered from the same APC.

In one embodiment the invention provides a method of stimulating andactivating antigen specific T cell expansion employing antigenpresentation on a first cell (or population of cells) and an artificialco-stimulatory factor which is a second distinct cell.

In one embodiment the engineered cell line, such as the aK562 cell lineis irradiated before use in the method of the present disclosure, forexample with a gamma radiation source of an X-ray source. The engineeredcell line, such as the aK562 cell line may be provided in a frozen formin which case irradiation of the cells may be performed after freezing.

In one embodiment the ratio of CTLs to co-stimulatory factor (forexample where the co-factor is a cell line) is in the ratio of 2:1 to1:10 respectively, such as 1:5.

Suitable ratios of CTLs:antibody:co-stimulatory cells are in the range1:1:0.2 to 1:1:10, such as 1:1:5. Advantageously, the method employingT-APCs and artificial co-stimulatory factors, such as engineered cells,may provide for improved levels of antigen specific T cell expansion incomparison to prior art methods.

The cytokine or cytokines employed in step a) and optionally step b)must be appropriate for stimulating and/or activating T cell growth ordifferentiation or perform some other useful function such as promotes Tcell survival or the like.

Cytokines that may be employed in the process of the current disclosureinclude IL-1, IL-2, IL-4, IL-6 IL-7, IL-12 and IL-15.

In one embodiment the cytokines employed in the process according to thepresent disclosure are independently selected from IL-4, IL-7 and IL-15,such as a combination thereof.

In one embodiment in step a) the cytokines employed are IL-4 and/orIL-7. Whilst not wishing to be bound by theory the inventors believethat these cytokines have a role to play in regards to frequency,repertoire and expansion of viral antigen specific cells.

In one embodiment if IL-2 is employed in step a) then it is added atabout day 3 or 4 of the culture and not at the outset.

The repertoire of T cells may be determined by ELISPOT analysis afterstimulation with peptide libraries aliquotted into pools such that eachpeptide is uniquely represented in two pools (Kern, F., N. Faulhaber, C.Frommel, E. Khatamzas, S. Prosch, C. Schonemann, I. Kretzschmar, R.Volkmer-Engert, H. D. Volk, and P. Reinke. 2000. Analysis of CD8 T cellreactivity to cytomegalovirus using protein-spanning pools ofoverlapping pentadecapeptides. Eur J Immunol. 30:1676-1682 andStraathof, K. C., A. M. Leen, E. L. Buza, G. Taylor, M. H. Huls, H. E.Heslop, C. M. Rooney, and C. M. Bollard. 2005. Characterization oflatent membrane protein 2 specificity in CTL lines from patients withEBV-positive nasopharyngeal carcinoma and lymphoma. J. Immunol.175:4137-4147).

In one embodiment in step b) the cytokine employed is IL-15. Whilst notwishing to be bound by theory it is believed by the inventors that theIL-15 has a pro-survival effect on the relevant T cell population. Inone embodiment the cytokine employed in step b) is IL-15.

Cytokines such as IL-15 may be replaced during culture e.g. twiceweekly.

In one embodiment only the particular cytokines described in any one ofthe embodiments herein are employed.

IL-12 has a role in ThI focusing and exogenous IL-12 may be omitted if abalanced Th1/Th2 is desired. In one embodiment the process of thepresent disclosure does not employ exogenous IL-12. However, in thecontext of the present T cell product a ThI response in the CD4+population is thought to be desirable.

The exogenous cytokines may be added at any stage of the process asappropriate, including concomitant addition when the cells aretransferred into the culture system or at the start of the given step.The latter applies to step a) and/or step b).

The presence of exogenous cytokines in step a) and/or step b) mayalternatively be added part way through the step, for example 1, 2 3, 4days or more after the step is initiated.

In one embodiment the process of the present disclosure is employed toprovide cell population comprising a CD4+ T cell population, for examplea Th1 population. A Thi population as employed herein is intended torefer to a CD4+ population wherein 5% of the cells or more, such as 10,20, 30, 40, 50, 60, 70, 80, 90% or more are classified as Th1.

Th1 cells, amongst other functions, generally maximize the killingefficacy of macrophages and the proliferation of cytotoxic CD8+ cells.

Memory T cells are a sub-category of Th1 cells.

In one embodiment the population of cells obtained from the processcomprise a sub-population of memory T cells. Whilst not wishing to bebound by theory we believe that a substantial portion of the T cellsobtained from the process will be derived from the memory portion of thestarting population.

In one embodiment IL-2 is not employed in step (a) because the expansionpromoted by this cytokine may be too non-specific and produce expansionof NK cells, T cells of unwanted function/specificity and T regulatorycells that may produce a certain amount of anergy in the cells. Thus inone embodiment the only IL-2 present in the culture is endogenous IL-2,i.e. it is secreted by the cells.

In one embodiment IL-2 is not employed in step (b) because it maypromote a more differentiated phenotype than IL-15

Anergy as employed herein is intended to refer to a lack ofresponsiveness of the cells to antigen stimulation. Anergy can bemeasured using a functional assay, for example interferon gammasecretion by the relevant cell population. A lower level of interferongamma secretion in comparison to full functional (non-anergic) cells maybe indicative of a degree of anergy. Of course the greater the degree ofanergy in the cells the lower the particular (marker) functionality willbe.

As descried above anergy in the context of the expanded product isintended to be a generic term that refers to reduced cell function inone or more relevant ways. The term includes cell exhaustion, forexample where the cells are no longer able to divide. The cell is thenreferred to as senescent. Cells stop dividing because the telomeres,protective bits of DNA on the end of a chromosome required forreplication, shorten with each copy, eventually being consumed.

It is thought that that PD-1 (programmed cell death protein 1; UniprotQ15116) which is expressed on the surface of cells, is a marker ofanergy. In one embodiment less than 10%, for example 9, 8, 7, 6, 5, 4,3, 2, 1, 0.5% or less of the relevant expanded T cell population expressPD-1, such as about 1%. In patients with high levels of anergy in the Tcells such as patient's with relapsed NPC the frequency ofLMP/EBNAI-specific T cells may be increased in the presence of blockingantibodies to PD-L1 or PD-1, i.e. which block signaling from the ligandbinding to the receptor.

This phenomenon was not found in healthy donors, suggesting that type 2latency antigen specific T cells might be anergic in NPC patients.

Target antigen as employed herein is intended to refer to the antigenwhich is employed to generate specificity in the T cells to thetherapeutic target, such as a particular virus, for example EBV. Thusthe cells infected by target virus or cancer cells will usually expressthe target antigen and hence will themselves become a target forclearance by the immune system.

In one embodiment the population of T cells expanded are a balance CD4+and CD8+ population, that is to say the cell population comprises bothCD4+ and CD8+ cells but not necessarily in equal amounts. Balanced inthe context of the present specification is employed to infer that oneof the populations CD4+ or CD8+ is not depleted during expansion.

The cell populations expanded using the process of the presentdisclosure comprises the desired T cell population and generally willnot consist only of the desired population. The final productadministered to the patient may include a number of other cells that theprocess did not target the expansion of. In one embodiment the desiredpopulation of CD4+ and CD8+ cells comprises about 60% or less of thetotal population of cells. Frequency of the cell populations may bemeasured employing a gamma-IFN ELISPOT assay or employing Multimer (e.g.Tetramer) staining both of which are known to persons skilled in theart.

In one embodiment the T cells population obtained from the process arediverse when analyzed by spectratyping, but without the emergence ofdominant clone. That is to say the T cell diversity in the startingsample is substantially represented in the expanded T cells, i.e. theexpansion is not generally providing a monoclonal or oligo clonal targetcell population.

A significant proportion for example 30% to 60% of the expanded cellswill generally express effector memory markers including CD27, CD28,CD62L and/or CD45RO.

It is expected that 50% or more, such as, 60, 70, 80, 90 or 95% of theantigen specific T cells according to the present disclosure may becapable of killing target cells expressing antigens that were used inthe expansion process.

We believe sufficient cells even for the highest cell doses required forthe treatment of patients can be prepared employing two stimulationsemploying methods of the present disclosure taking in the range of 16 to24 days of T cell culture compared to 28 to 63 days of LMP-T cellculture using Ad vectors and LCLs.

What is more the cells produced by the process of the present inventionmay be advantageous in that there are more specific, produce higherlevels of gamma interferon and/or express lower levels of anergy markerscompared to cells prepared by the prior art processes.

In one embodiment, anergic T cells from cancer patients may grow poorlyand require three stimulations. The third stimulation might includeadditional stimulatory components. Since at this time the majority ofcells should be specific, there is not a concern about expansion ofunwanted cells. In one embodiment the third stimulation culture willcomprise (1) peptide-pulsed, irradiated autologous activated T cells,(2) irradiated costimulatory cell line such as aK562 cells, (3)irradiated PBMCs from blood bank approved allogeneic donors and (4)submitogenic doses of anti-CD3; 1 to 100 ng per mL for example 50 ng perml.

In one embodiment one or more tests are performed to establish thesuitability of the cells for use in therapy, for example an interferongamma ICS (intracellular cytokine staining) and/or an interferon gammaELISPOT assay.

In one embodiment the suitability testing is combined in a single flowcytometry assay.

In one embodiment a ⁵¹Cr release assay for non-specific killing isperformed, but this will generally not form part of the suitabilityassays.

In one embodiment batch release testing is set out in Table 1:

TEST Specification 1. Viability Dye exclusion or flow cytometry >70%Viable 2. Purity Flow cytometry >80% CD3+ 3. Safety 3.1 Endotoxin (LALassay) ≤5.0 EU/ml 3.2 Bacterial sterility Aerobic Bacteria - BactecNegative Anaerobic Bacteria - Bactec Negative Fungal contaminants -Bactec Negative 3.3 Mycoplasma PCR Assay Negative 3.4 Non-specifickilling* <10% killing target cells at 20:1 E:T ratio 4. Identity HLAclass I antigen identical with patient/ donor 5. Potency 5.1 IFN- y +cells >3 standard deviations greater response to stimu- lation withspecific peptides than to control stimulation 6. Dosing CD3+ number 1-10× 10⁷ cells per m² or 2-20 × 10⁵ per kg BW

The process of the present disclosure may be performed in a well orcontainers but most suitably is performed in a gas-permeable system.Vessel as employed herein is intended to refer to any type of containersuitable for retaining the cells, media etc., for example a bag, such asan infusion type bag (provided with a gas permeable portion) or a rigidvessel, such as the GRex™ system. The gas permeable layer facilitatesrapid expansion of cells and minimizes the number of media changesrequired.

In one embodiment step a) is performed in a GRex™ 10 system. The GRex™10 system is suitable for culturing up to 1×10⁵ T cells.

In one embodiment step b) is performed in a GRex™ 100 system.

WO 2005/035728 incorporated herein by reference describes how to preparea gas permeable vessel. In one embodiment silicone gas permeablematerial is employed.

In one embodiment the system employed is a GRex™ system from WilsonWoolf. In one embodiment the system is adapted to provide asepticpreparation as described in U.S. provisional application Ser. No.61/550,246 incorporated herein by reference.

Generally the system is seeded with about 0.5 million cells per cm² ofsurface area. In a GRex-10 with a surface area of 10 cm², a minimum of 5million and up to 20 million cells would be seeded.

In one embodiment in step (a) 20 million cells might be seeded in aGRex-10. Within PBMC, less than 1% of cells are specific for thepeptides, so at most 0.2 million specific cells are seeded. Theremaining PBMCs act as feeder cells.

In one embodiment in step (b) 50 million irradiated a K562 cells plus 10million peptide-pulsed activated and irradiated T cells and 10 millioneffector T cells would be seeded into a GRex-100 (100 cm²), In this caseonly the effector T cells will proliferate.

In one embodiment the present disclosure extends to the cell compositionobtained directly from the process.

In one embodiment the process according to the present disclosuregenerates sufficient CD3+ cells to provide at least two individual dosesfor a patient.

The invention also extends to compositions with the same desirablecharacteristics of the cell populations prepared by the methodsdisclosed herein.

Thus in one aspect the present disclosure provides an autologous T cellpopulation expanded in vitro to contain a population of T cells specificto a target antigen, such as a virus, wherein the population issubstantially free of target virus contamination and responses to viralvectors.

The present process also relates to the preparation of dendritic cellspulsed with peptide mixes and the cell populations obtained therefrom,for use in the expansion of antigen specific T cell populations,particularly as described herein.

The present process also relates to the process of preparing T-APCspulsed with peptide mixes and the cell populations obtained therefrom.

The present disclosure also relates to autologous T cell populationsdescribed herein, for example comprising a population of CD4+ and CD8+antigen specific T cells, wherein the antigen is associated with virusinfected cells or cancer cells.

In one or more embodiments the cell populations according to the presentdisclosure have one or more advantageous properties in comparison tocells prepared by the prior art method.

In one embodiment the average cell diameter of cells in the relevant Tcell population is 10 to 14 μM. Advantageously the cells cultures of thepresent invention produce generally low toxicity after infusion, forexample are associated with few toxicity intolerance responses, forexample inflammatory responses, cell damage, flu like symptom, nausea,hair loss or the like.

The cell populations according to the present disclosure also provideadvantageous properties, for example high levels of interferon gammaexpression.

High levels of interferon gamma expression as employed herein isintended to refer to the fact that on average cell populations preparedby the current method may express higher levels of interferon gamma thancells prepared by prior art methods and certainly express higher levelsof interferon gamma than the original anergic cells obtained from thepatient.

In one embodiment the cells of the present disclosure show enhancedantigen specificity, for example in an assay disclosed herein, forexample the cells of the present disclosure contain a higher frequencyof cells that secrete cytokines in response to stimulation with theantigens in comparison to cells prepared by a prior art method. Thisfigure will usually be a mean which is a per cell value derived from thevalues obtained for the population and divided by the number of cellspresent.

In one embodiment the cell populations of the present disclosure showcomparable avidity (not significantly different) to cell populationsprepared by a prior art method.

To determine avidity, autologous activated T cells may be pulsed withdilutions of peptide, labelled with ⁵¹chromium and used as targets in astandard cytotoxicity assay. The most avid T cells are those that killtarget cells pulsed with the lowest concentration of peptide.Alternatively IFN gamma production can be measures using an ELISPOTassay with dilutions of peptide.

In one embodiment the cell populations of the present disclosure showincreased ability to kill target cells in comparison to cell populationsprepared by a prior art method.

In one embodiment the T cell populations provided by the presentdisclosure are effective in expanding in vivo to provide an appropriateimmune response to cells infected by a target virus and/or cancer cellsassociated with a target virus.

The present invention also extends to compositions comprising theautologous T cell populations according to the invention. Thesecompositions may comprise a diluent, carrier, stabilizer, surfactant, pHadjustment or any other pharmaceutically acceptable excipient added tothe cell population after the main process steps. An excipient willgenerally have a function of stabilizing the formulation, prolonginghalf-life, rendering the composition more compatible with the in vivosystem of the patient or the like.

In one embodiment a protein stabilizing agent is added to the cellculture after manufacturing, for example albumin, in particular humanserum album, which may act as a stabilizing agent. The amounts albuminemployed in the formulation may be 10 to 50% w/w, such as about 12.5%.w/w.

In one embodiment the formulation also contains a cryopreservative, suchas DMSO. The quantity of DMSO is generally 20% or less such as about 12%in particular 10% w/w.

In embodiment the process of the present invention comprises the furtherstep of preparing a pharmaceutical formulation by adding apharmaceutically acceptable excipient, in particular an excipient asdescribed herein, for example diluent, stabilizer and/or preservative.

Excipient as employed herein is a generic term to cover all ingredientsadded to the T cell population that do not have a biological orphysiological function.

Once the final formulation has been prepared it will be filled into asuitable container, for example an infusion bag or cryovial.

In one embodiment the process according to the present disclosurecomprises the further step of filling the T cell population orpharmaceutical formulation thereof into a suitable container, such as aninfusion bag and sealing the same.

In one embodiment the container filled with the T cell population of thepresent disclosure or a pharmaceutical composition comprising the sameis frozen for storage and transport, for example is store at about −135°C., for example in the vapor phase of liquid nitrogen.

In one embodiment the process of the present disclosure comprises thefurther step of freezing the T cell population of the present disclosureor a pharmaceutical composition comprising the same. In one embodimentthe “product” is frozen by a controlled rate freezing process, forexample reducing the temperature by 1° C. per minute to ensure thecrystals formed are small and do not disrupt the cell structure. Thisprocess may be continued until the sample has reached about −100° C.

A product according to the present disclosure is intended to refer to acultured cell population of the present disclosure or a pharmaceuticalcomposition comprising the same.

In one embodiment the product is transferred, shipped, transported in afrozen form to the patient's location.

In one embodiment the product according to the present disclosure isprovided in a form suitable for parenteral administration, for exampleinfusion, slow injection or bolus injection. In one embodiment theformulation is provided in a form suitable for intravenous infusion.

In one aspect the present disclosure provides a method of transporting aproduct according to the present disclosure, from the place ofmanufacture, or a convenient collection point to the vicinity of theintended patient, for example where the T cell product is stored below0° C., such as −135° C. during transit.

In one embodiment the temperature fluctuations of the T cell product aremonitored during storage and/or transport.

In one embodiment there is provided a product of the present disclosurefor use in treatment, for example in the treatment of a viral associateddisease or malignancy, such as EBV infection, CMV infection, adenovirusinfection, HIV infection, hepatitis C or B infection, parvovirusinfection, influenza virus infection, or a cancer from viral origin, forexample EBV-associated lymphoma or carcinoma, HHV8-associated sarcoma,papillomavirus-associated carcinoma or SV40-associated cancers.

Other viral morbidities include varicella zoster virus infection,vaccinia virus infection and complications of either of the same.

In one embodiment the treatment is of an immunosuppressed patient.

In one embodiment, the patient is not immune-compromised.

In one embodiment there is a provided a method of treating a patientwith a product according to the present disclosure comprising the stepof administering a therapeutically effective amount of product definedherein.

Therapeutically effective amount, does not necessarily mean an amountthat is immediately therapeutically effective but includes a dose whichis capable for expansion in vivo (after administration) to provide atherapeutic effect.

Thus there is provided a method of administering to a patient atherapeutically effective amount which is a sub-therapeutic dose ofexpanded T cells which are capable for expansion in vivo to provide thedesired therapeutic effect, for example.

In one embodiment the antigen specific T cell population produced isspecific to EBV virus and prevents, ameliorates or eliminates cellsinfected with EBV and/or clinical pathologies associated therewith, forexample EBV associated cancers.

Symptoms of infection include fever, sore throat, and swollen lymphglands. Sometimes, a swollen spleen or liver involvement may develop.Heart problems or involvement of the central nervous system occurs onlyrarely. EBV remains dormant or latent in a few cells in blood for therest of the person's life and can be reactivated, for example inimmunosuppressed patients when immune controls are reduced or absent.Whilst the infection is not fatal in individuals with a healthy immunesystem, the infection can lead to severe complications and death inimmunosuppressed patients.

EBV is best known as the cause of infectious mononucleosis. It is alsoassociated with particular forms of cancer, particularly Hodgkin'slymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngealcarcinoma, gastric carcinoma and central nervous system lymphomasassociated with HIV. Finally, there is evidence that infection with thevirus is associated with a higher risk of certain autoimmune diseases,especially dermatomyositis, systemic lupus erythematosus, rheumatoidarthritis, Sjögren's syndrome, and multiple sclerosis.

In one embodiment the target patient population for treatment hasnasopharyngeal carcinoma. In one embodiment the target patientpopulation for treatment has gastric carcinoma.

Thus T cell populations specific to EBV according to the presentdisclosure can be employed in the treatment of prophylaxis of one ormore of the above conditions.

In one embodiment there is provided a method of treatment or prophylaxisof a varicella zoster virus infection and/or complications associatedtherewith, including encephalitis, pneumonia, posthereptic neuralgia,shingles, zoster multiplex, myelitis, herpes opthalmicus and zoster sineherpete, comprising administering a therapeutically effective amount ofa T cell population comprising T cells specific to VZV, according to thepresent disclosure.

In one embodiment there is provided a method of treatment or prophylaxisof a vaccinia virus infection and/or complications associated therewith.

In one embodiment a dose of at least 1×10⁷ cells per m² or 2×10⁵ per kgis employed.

In one embodiment a first dose of about 2×10⁷ per m² and a second doseof 2×10⁷ or 1×10⁸T cells per m² is employed.

In one embodiment the present disclosure provides use of a peptide mixcovering a viral or cancer antigen(s) for generating autologousdendritic cells suitable for use in expansion of autologous antigenspecific T cells.

In one embodiment the present disclosure provides use of a peptide mixcovering a viral or cancer antigen(s) for generating autologous T-APCssuitable for use in the expansion of antigen specific T cells. In oneembodiment the present disclosure provides use of aK562 cells or otherartificial co-stimulatory factory for use as a co-stimulatory factorwithout concomitantly presenting antigen thereon in the expansion ofantigen specific T cells (in particular autologous T cell expansion),for example employed in conjunction with T-APCs for expansion of antigenspecific T cells, in particular antigen specific T cells, such asautologous antigen specific T cells.

In one embodiment there is provided a kit comprising a peptide mixcovering a viral or cancer antigen(s) and an artificial co-stimulatoryfactor such as an aK562 cell, in particular for use in T cell expansion,such as autologous antigen specific T cell expansion. In one embodimentthe kit further comprises IL-15.

In one embodiment the kit comprising a peptide mix covering a viral orcancer antigen(s) and IL-4 and IL-7 for use the expansion of antigenspecific T cells, for example autologous antigen specific T cells.

PROTOCOLS AND EXAMPLES Abbreviations

-   APCs Antigen presenting cells-   ATC Activated T cell-   CTLs Cytolytic T lymphocytes-   DCs dendritic cells-   EBV Epstein-Barr Virus (a virus from the Herpes family of viruses)-   LCL lymphoblastoid cell line-   EBV-LCL lymphoblastoid cell line infected with EBV-   LMP1 Latent Membrane Protein 1 Uniprot number P03230-   LMP2 Latent Membrane Protein 2 Uniprot number Q1HVJ2-   BARF1 protein encoded by the BamHI rightward reading frame uniprot    number-   EBNA1 EBV nuclear antigen 1 Uniprot number P03211-   PBMC peripheral blood mononuclear cell-   CMV Cytomegalovirus    Protocol 1: Processing of Sample to Obtain PBMCs

The steps below should be performed in a certified biological safetycabinet, using aseptic techniques and following universal precautions.

A blood sample or an apheresis sample or a buffy coat sample (which isthe fraction of an anti-coagulated blood sample after density gradientcentrifugation that contains most of the white blood cells andplatelets) is diluted with an equal volume of Dulbecco's PhosphateBuffered Saline or RPMI medium (for example RPMI 1640 available fromLife Technologies), at ambient (room) temperature.

In a 50 ml centrifuge tube, 15 ml Lympho-prep is carefully overlaid withapproximately 30 ml of diluted blood. This step can be adjusted toutilize all the available cells.

The material is centrifuged at 400×G for about 40 minutes at ambienttemperature.

Aliquots, for example 3×1 ml plasma aliquots may be stored at −80° C.

The PBMCs interface is harvested into an equal volume of Dulbecco'sPhosphate Buffered Saline or RPMI medium (for example RPMI 1640available from Life Technologies). Centrifuge at about 450×G for about10 minutes at room temperature and then aspirate supernatant. The pelletobtained should be loosened and re-suspended in about 20 ml Dulbecco'sPhosphate Buffered Saline or RPMI medium (for example RPMI 1640available from Life Technologies).

If the process is performed multiple times then the cells may becombined in a single centrifuge tube and thoroughly re-suspended. Thecells will generally be counted by removing 20 μl of cells and adding 20μl of 50% red cell lysis buffer and counted using a hemacytometer inaccordance with the manufactures instructions.

The PBMCs may be used in the expansion antigen specific T cells,expansion of CD3 and CD28 activated T cells and in the preparation ofdendritic cells.

NOTES: Since the PBMCs cells prepared are ultimately intended forinfusion into patients it is essential to adhere to procedures for theidentification and handling of patient samples. Only one patient'ssample should be handled at a time. Generally the number of PBMCsrecovered will be in the range 0.5 to 2.0×10⁶ PBMCs per ml of blood. Upto 1×10⁹ may be recovered from a buffy coat.

REFERENCES

-   Sili U et al Large-scale expansion of dendritic cell-primed    polyclonal human cytotoxic T-lymphocyte lines using lymphoblastoid    cells for adoptive immunotherapy. J. Immuother. 2003 May-June:    26(3): 241-56-   Leen A M et al Contact-activated monocytes: efficient antigen    presenting cells for the stimulation of antigen-specific T cells. J    Immunother. 2007 January: 30(1): 96-107.-   Bollard C M et al The generation and characterization of    LMP2-specific CTL for use as adoptive transfer from patients with    relapsed EBV-positive Hodgkin disease J. Immunother.    Protocol 2: Generation of Dendritic Cells

Dendritic cells can be differentiated from adherent of CD14-selected PBmononuclear cells (PBMC) by culture in GM-CSF and IL-4. The dendriticcells can then be matured using GM-CSF, IL-4, IL-1β, IL-6, TNF-α andPGE-1 or PGE-2 (PGE=Prostaglandin E). The dendritic cells loaded withpeptides can present the peptides on HLA class I and class II moleculesto antigen-specific T cells via the TCR.

Preparation of Adherent PBMCs—Dilute heparinized peripheral blood, forexample 30 ml, in an equal volume of Dulbecco's Phosphate BufferedSaline or RPMI medium (for example RPMI 1640 available from LifeTechnologies), at ambient (room) temperature.

Alternatively thaw previously frozen PBMC, wash twice in CellGenix DCmedium, and count the cells.

In a 50 ml centrifuge tube, 15 ml Lymphoprep is carefully overlayed withapproximately 30 ml of diluted blood. This step can be adjusted toutilize all the available cells.

The material is centrifuged at 400×G for about 40 minutes at ambienttemperature.

The PBMCs interface is harvested into an equal volume of Dulbecco'sPhosphate Buffered Saline or RPMI medium (for example RPMI 1640available from Life Technologies). Centrifuge at about 450×G for about10 minutes at room temperature and then aspirate supernatant. The pelletobtained should be loosened and re-suspended in about 20 ml Dulbecco'sPhosphate Buffered Solution.

The above steps are the same as Example and then the material is thencentrifuged at 400×G for about 5 minutes at room temperature. Thesupernatant is then removed and the pellet is re-suspended in 20 ml ofDC medium.

Count cells as defined in Example 1. If the concentration is greaterthan 5×10⁶ per ml adjust to 5×10⁶ per ml by adding CellGenix DC medium.If the concentration is less than 5×10⁶ per ml the pellet and re-suspendat 5×10⁶ per ml in CellGenix DC medium.

Transfer 10 ml of cells per 75 cm² flask or 2 ml of cells/well of a 6well plate.

Transfer to 37° C. and 5% carbon dioxide for about 2 hours. Rinsing theflask three times 10 mls of Dulbecco's Phosphate Buffered Saline or RPMImedium combining the supernatants contain thing the PBMC non-adherentfraction.

For a T-75 culture flask (available from Falcon) add 10 ml of DC culturemedium containing 1000 units per ml of IL-4 and 800 units per ml ofGM-CSF to the adherent cells.

For a 6-well plate add 2 mls of DC culture medium containing 1000 unitsper ml of IL-4 and 800 units per ml of GM-CSF to the adherent cells.

Transfer the flasks or plated to an incubator at 37° C. and 5% carbondioxide.

If not previously cryopreserved, non-adherent cells may be cryopreservedfor future use in the preparation of responder T cells.

On day 3 or 4 add 1000 units per ml of IL-4 and 800 units per ml ofGM-CSF.

A summary of the cytokines added are provided in Table 1:

Final Volume to be Cytokine Concentration Stock Added per ml GM-CSF 800U/ml 2,800 U/ml 3.8 μl IL-4 1000 U/ml 4000/μl 33 μl TNF-α 10 ng/ml1μ1/ml Dilute 13 μl Diluted 1:100 (10 ng/ml) solution PGE-1 1 μg/ml 0.5g/ml 2.7 μl IL-1β 10 ng/ml 10 ng/μl 10 μl IL-6 100 ng/ml 10 ng/μl 10 μl

The culture is then incubated for about 2 days at 37° C. and 5% carbondioxide after which the dendritic cells are ready of preparation asstimulators for autologous PBMC.

The dendritic cells are harvested and counted. Note that cells are lostwhen washed several times, therefore the wash steps can be omitted whendendritic cell numbers are limiting. Dendritic cells do not divide andthe omission of irradiation is not important.

If greater than 5×10⁵ transduced dendritic cells are recovered sendapproximately 10⁵ for phenotyping along with non-transduced dendriticcells if the latter are available.

If less than 5×10⁵ transduced dendritic cells are recovered then proceedto peptide loading.

The supernatant is aspirated after centrifugation and the 5 μl (5 ng) ofeach peptide library (i.e. for each antigen) per 1×10⁶ dendritic cellsis added. The mixture is then incubated for 30 to 60 minutes in 5%carbon dioxide incubator and then irradiated at 30 Grays.

The cells are re-suspended in 20 mls of medium and centrifuged for about5 minutes at 400×G. Re-suspend the cells at 10⁵ per ml with CTL culturemedium. The dendritic cells are now ready for use as stimulators forPBMCs.

As with Example 1 because these cells are to be infused into patientsappropriate procedures must be followed.

Recovery of total dendritic cells should be 1 to 8% of the startingpopulation.

REFERENCES

-   Gahn B et al Adenoviral Gene Transfer into Dendritic Cells    Efficiently Amplifies the Immune Response to the LMP2A-Antigen; a    potential treatment strategy for EBV virus-positive Hodgkin's    lymphoma. Int. J. Cancer 2001 Sep. 1; 93(5): 706-13-   Bollard C M et al The generation and characterization of    LMP2-specific CTLs for use in adoptive transfer from patients with    relapsed EBV-positive Hodgkin disease J. Immunother (1997). 2004    July-August; 27(4):317-327-   Gottschalk S et al Generating CTLs against subdominant Epstein-Barr    virus LMP1 antigen for the Blood 2003 Mar. 1; 101(5): 1905-12.    Protocol 3: Generation of T-APCs    1. Coat non tissue culture treated 24 well plates or T-75 non tissue    culture treated flasks with CD3 and CD28 antibodies (Miltenyi)    1.1. Calculate the number of wells/flasks to be coated based on the    PBMC number to be plated at 1×106 cells per well or 30-50×106 cells    per T-75 flask    1.2. For wells, plate 0.5 ml of H₂O containing 1 ng CD3 (5 μl/ml of    H₂O of 0.2 mg/ml stock) and 1 g CD28 (2 L/ml of H₂O of 0.5 mg/ml    stock) per well.    1.3. For T-75 flasks, after prewashing with 18 ml of H₂O; use 90 C51    CD3 (stock 0.2 mg/ml) and 36 μl CD28 (stock 0.5 mg/ml) in 18 mls H₂O    per flask.    1.4. Incubate for at least 3 hours in 37° C. incubator.    1.5. Optional: CD3-CD28 coated plate can be incubated overnight at    4° C.    2. Wash wells or flasks    2.1. Remove CD3/28 solution and rinse once with 1 mL of PBS or    medium per well or 10 mls per T-75 flask    3. Re-suspend PBMCs at 2×10⁶ in 30 mL of T cell medium (10% human AB    serum, 45% RPMI 1640 (or advanced RPMI), 45% EHAA and 200 mM    L-glutamine)    3.1. Aliquot 2 mls per CD3/28-coated well or 15 to 25 mls to a T-75    flask    3.2. Transfer to incubator for 2 days with flask lying flat.    4. On day 2 add 100 units per mL of IL-2.    5. On day 4 to 5 cells re-suspend cells and count.    6. Split cells according to the cell number obtained    6.1. 5×106 cells per GRex 10    6.2. 5×107 per GRex 100.    7. 3 to 4 days later, harvest cells and count    7.1. Either further expand as in step 4    7.2. Or cryopreserve    8. Cryopreservation    8.1. Centrifuge cells and aspirate supernatant    8.2. Flick pellet to re-suspend and transfer to ice for 10 to 30    minutes    8.3. Re-suspend at 5×10⁶ to 10×10⁶ cells per ml of ice cold    cryopreservation medium (10% DMSO, 50% human AB serum, 40% RPMI    1640)    8.4. Transfer to cryocontainers (cryobags or cryovials)    8.5. Freeze in freezing containers at ˜1° C. per minute for at least    90 minutes, then transfer to liquid nitrogen    9. Prior to use at antigen-presenting cells, T cells must be    re-stimulated on CD3/28 antibody-coated plates as in step 3, for 2    to 4 days to upregulate co-stimulatory molecules    9.1. This step is critical as if T cells have a resting phenotype,    they may induce T regulatory cells    Protocol 4: Expansion of Autologous Antigen Specific T Cells Using a    Preferred Embodiment of the Invention    1. Re-suspend PBMCs at 2×10⁶ per mL of T cells medium containing    IL-4 and IL-7.    T cell medium is 10% human AB serum, 45% Advanced RPMI, 45% EHAA and    200 mM L-glutamine. IL-4 is added at 20 ng per mL (10 ng/ml final    dilution) and IL7 at 3332 units per mL (1666 U/ml final dilution)    2. Re-suspend peptide-coated dendritic cells at 1×10⁵ to 2×10⁵ per    ml    3. Mix PBMCs and DCs at a 1:1 ratio    4. Transfer 20 mls per GRex-10 (20×10⁶ PBMCs) and add 10 mls    additional medium containing IL-4 (1666 units per mL) and IL-7 (long    per ml). Transfer to incubator for 7 days    5. On day 7 remove 20 mls medium    Re-suspend cells and count.    If >50×10⁶ cells, split between two GRex-10s. If <50×10⁶ leave in    one GRex-10.    Add medium containing IL-4 and IL-7 to 30 mls in each GRex-10 (final    concentration of IL-4 is 1666 units per mL and of IL-7 is 10 ng per    ml)    Transfer to incubator for 2 to 3 more days    6. On Day 9 or 10 re-stimulate with peptide-coated T-APCs and aK562    cells    Harvest responder T cells from GRex and count    Re-suspend at 1×10⁶ cells per ml    Transfer to Incubator    6.1 Prepare aK562-CS cells to provide a 5:1 aK562-CS to T cell ratio    6.1.1. Irradiate aK562-CS cells with 100 GY (Grays)    6.1.2. Centrifuge for 5 minutes and 400 G    6.1.3. Re-suspend in complete T cell medium    6.1.4. Count and estimate the number of aK562-CS cells required    6.1.4.1. The number of responder T cells×5    6.2. Prepare peptide-pulsed, irradiated, activated autologous T    cells (T-APCs)    6.2.1. Autologous T cells (ATCs) should have been stimulated or    re-stimulated with CD3/28 two to 4 days before use    6.2.2. Harvest sufficient number of ATCs for stimulation plus ˜30%    to account for loss during processing    6.2.3. Centrifuge cells at 400 G for 5 minutes and aspirate    supernatant    6.2.4. Loosen cell pellet by finger-flicking    6.2.5. Add long of each peptide per 10×10⁶ ATCs    6.2.6. Incubate at 37° C. in 5% CO₂ in air for 30 to 90 minutes    6.2.7. Re-suspend in ^(˜)20 mL medium    6.2.8. Irradiate 30 GY    6.2.9. Centrifuge 400 G for 5 minutes and aspirate supernatant    6.2.10. Re-suspend at 10⁶ cell per mL    6.3. Combine 1×10⁷ responder T cells with 1×10⁷ T-APCs and 5×10⁷    irradiated aK562-CS cells per GRex-100    6.3.1. Add medium to 400 mL    6.3.2. Add 10 ng per mL IL-7 (2 mg) and 6.6×10⁶ units of IL-4    6.3.3. Transfer to incubator for 3 to 4 days    7. Add IL-2 (50 to 100 units per mL) or IL-15 10 ng per mL    7.1. Return to culture for 3 to 4 days    7.2. Add cytokines every 3 to 4 days    8. Measure glucose from day 7    8.1. Remove 1 drop of medium and test on standard hand held    glucometer    8.2. Glucose levels of less than 100 should trigger a change of    medium/cytokines    8.3. Responder T cells should be cryopreserved when sufficient    number have been obtained    9. A third stimulation (steps 7, 8 and 9) can be performed if    insufficient cells are obtained

Example 1: Generation of EBNA1, LMP1 & LMP2 Specific T Cells for HealthyDonors and Patients with Nasopharyngeal Carcinoma and Lymphoma

Sub Experiment: Generation of EBNA1, LMP1 & LMP2 Specific T Cells forLymphoma Patient 1.

D1 Coating OKT3/CD28 Plate

Prepared OKT3 and CD28 antibodies by adding 5 ug each of OKT3 and CD28antibodies to 5 mL of sterile water. Added 0.5 mL of this mixture intoeach well of a non-tissue culture treated 24 well plate (24-w-p).Incubated at 4° C. overnight. Note: this is culture day (−)8.D2 Generation of dendritic cells from frozen PBMCs by adherence &generation of OKT3 blasts from non-adherent PBMC population—culture day(−)7Lymphoma patient 1 peripheral blood mononuclear cells (PBMCs) frozenstock taken out from GMP bank and follow up samples with permission ofPI (Cath Bollard): 4 vials of PBMCs, all frozen between February andMarch 2010, 5 million each vial, total 20 million. PBMCs were thawed outin 2 vials of 40 mL warm CellGenix media and spun down. PBMC count ofpatient 1: 22.5 million Spun down patient 1 PBMCs and re-suspended in atotal of 6 mL of warm CellGenix media, then plated PBMC out in 3 wellsof a 24-w-p with 2 mL per well. After 3 hours the non-adherent portionwas gently washed off with sterile PBS twice and media was replaced withCellGenix with IL4 and GM-CSF. Incubated at 37° C. The non-adherentportion was used to generate OKT3 blasts. OKT3 blast generation:A plate with OKT3 and CD28 coated the day before and stored at 4° C. waswashed once with 0.5 mL of T cell media per well. Non-adherent portionof PBMCs was plated out into ^(˜)10 wells. Incubated at 37° C.D3 Fed OKT3 blasts with IL2 by replacing ½ of the media in the well withCTL Media with 100 units of IL2/mL to make final culture concentrationof 50 units/mLD4 Fed DCs with IL4 & GMP-CSF by replacing 1 mL of media per well withfresh media with IL4 & GMP-CSFMoved OKT3 blasts into a new tissue culture treated 24-w-p. Incubated at37° C.D7 Transduced 1 well of each patient DC with Ad-LMP1-LMP2 by harvestingcells with scraping, counted, spun down then added Ad-LMP1-LMP2 at MOIof 5000.Patient 1: 0.5 million DC, thus added 0.5 u1 of virus at 5×10{circumflexover ( )}12 vp/mL concentration Flicked tubes every ˜15 minutes whileincubating at 37[1*]° C. for 1.5 hrs. Resuspended cells back in mediacontaining GM-CSF, IL4, IL1b, IL6, TNFa, and PGE2 and plated out backinto 1 well each of a 24-2-p with 2 mL of media.For the condition without Adenoviral vector, ½ media was taken off, andthen CellGenix media with 2× of GMP-CSF, 1L4, IL1b, IL6, TNFa and PGE2was added back into the wells. Incubated at 37° C.Continued feeding OKT3 blasts by replacing media or splitting cells intoextra well.D9 DO Setting up first stimulation.Harvested and counted dendritic cells:Patient 1: Non-transduced DCs: 0.2 million; transduced DC: 0.05 millionRemoved a frozen vial of patient 1 PBMCs from GMP (cells frozen in 2009)to be stimulated, then thawed cells using warm CTL media with 30% FBS:Patient 1: 1 vial of frozen PBMCs at 10 million per vial, recovered 8millionPulsing of DC and whole PBMCs with pepmixes:

Diluted EBNA1, LMP1 and LMP2 pepmixes by adding 1 u1 of stock pepmixsolution (in DMSO) each into 200 u1 of sterile PBS.

Spun down all non-transduced DC from both donors and aspirated all but˜50 u1 of media. Loosened pellets by flicking the tubes. Added ˜10 u1 ofdiluted pepmix to the pellets, incubated at 37[*]° C. for 30 min withoccasional flicking.

3 million of PBMCs from donor 1 were taken to a new clean tube each,spun down, and all but ˜50 u1 of media aspirated. Added ˜3 ul of dilutedpepmix into the pellet. Incubated at 37[*]° C. for 30 min withoccasional flicking.

Setting up T cell culture Day 0 for patient 1:

Current GMP condition: Spun down and re-suspended DCs into 0.5 mL ofmedia after incubation; took out 1 million of PBMCs, spun down andre-suspended in 0.5 mL of complete CTL media (2 million per mL). Addedboth into a well of a 48-w-p. Added 5 ug of IL15 to make finalconcentration of 5 ng/ml. DC:PBMC ratio of this condition was 1:20.Incubated at 37° C. Condition DC(px): Washed DC pulsed with pepmixeswith 20 mL of PBS. Re-suspended with 2 mL of complete CTL media, platedout 1 mL per well of a 24-w-p. Took out 4 million of PBMCs, spun downand re-suspended at 2 million per mL. Added 1L4 & IL7 at 2×concentration (20 ng/mL) to PBMCs. Added 1 mL into each well thatalready had DC to bring final concentration of IL4 and 117 down to 10ng/mL. DC:PBMC ratio of this condition was 1:20. Incubated at 37° C.Condition Px: Re-suspended PBMC pellet in 2 mL of complete CTL media,added ILA and 1L7 at 10 ng/mL, plated out into 1 well of a 24-w-p.Incubated at 37° C.D10 Patient 1 OKT3 blasts expanded to 2 full 24-w-p. Count: 65 million.Frozen down in 4 vials with each vial at ˜15 million cells.D15 Coating OKT3/CD28 platePrepared OKT3 and CD28 antibodies by adding 5 ug each of OKT3 and CD28antibodies to 5 mL of sterile water. Added 0.5 mL of this mixture intoeach well of a non-tissue culture treated 24 well plate (24-w-p).Incubated at 4° C. overnight.D16 Transduced LCL with Ad-LMP1-LMP2˜3 million of LCL from donor 1 culture was taken out, spun down, thenadded 3 u1 of virus at concentration of 5×10{circumflex over ( )}12vp/mL for MOI of 5000. Incubated at 37[*]° C. for 1.5 hr with intervalflicking of tube. After incubation re-suspended cells in ˜5 mL ofcomplete RPMI. Incubated at 37° C.Activated OKT3 blasts:Thawed out a vial each of OKT3 blasts from donor 1 at 10 million pervial in warm CTL media with 30% FBS. Spun down cells and re-suspended in20 mL of CTL media. Washed a plate of OKT3 & CD28 coated with CTL media,then plated cells out into 10 wells of a 24-w-p. Incubated at 37° C.D18 Culture day 10Harvested and counted day 10 T cells from patient 1:Condition GMP: 2.13 millionCondition DC(px): 2.4 millionCondition Px: 3.04 millionHarvested patient 1 OKT3 blasts and irradiated at 30 Gy. Count: 13.4millionHarvested aK562cs and irradiated at 100 Gy. Count: 4.4 million. Washed,spun down and re-suspended in 11 mL for a cell concentration of 0.4million per mLHarvested patient 1 transduced LCL, irradiated at 40 Gy. Count: 5.7millionBecause we didn't have enough cells for testing antigen specificity byELISpot or phenotyping for lymphocyte subtypes, we only stimulated thesecultures.Second stimulation:Pulsing of OKT3 blasts with pepmixes:

-   -   Diluted EBNA1, LM P1 and LMP2 pepmixes by adding 1 ul of stock        pepmix solution (in DMSO) each into 200 ul of sterile PBS.    -   Spun down OKT3 blasts and aspirated all but ⁻50 ul of media.        Loosened pellets by flicking the tubes. Added ⁻20 ul of diluted        pepmix to the pellets, incubated at 37[*]° C. for 30 min with        occasional flicking. Washed with 20 mL of PBS, spun down.    -   Re-suspended OKT3 blasts at concentration of 1 million per mL        Condition GMP:        Spun down LCL and re-suspended in 14.25 mL of CTL media for        concentration of 0.4 million per mL. Plated out 0.5 mL or 0.2        million LCL into 4 wells of a 24-w-p.        Spun down D9 T cells for GMP condition, re-suspended in 4 mL for        approximately 0.6 million of T cells per well and added 1 mL to        each of the 4 wells with LCL. T cell to LCL ratio was about 3:1.        Incubated at 37° C.        Condition DC(px):        Spun down DC(px) day 9 T cells, re-suspended in 4 mL of T cell        media with 2×IL4 and IL7 concentration at 20 ng/mL. Plated out 1        mL per well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3        blasts, then added 0.5 mL of aK562cs into each of the well to        make a final volume of 2 mL per well, with T cell: OKT3        blasts:aK562cs ratio of 1:1:1 (low ratio of aK562cs due to low        total aK562cs available). Incubated at 37° C.        Condition Px:        Spun down Px day 9 T cells, re-suspended in 6 mL of T cell media        with 2×IL4 and IL7 concentration at 20 ng/mL. Plated out 1 mL        per well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3 blasts,        then added 0.5 mL of aK562cs into each of the well to make a        final volume of 2 mL per well, with T cell: OKT3 blasts:aK562cs        ratio of 1:1:1 (low ratio of aK562cs due to low total aK562cs        available). Incubated at 37° C.        D21 Added 1L2 to culture day 14 by replacing 1 mL of media in        each well with 1 mL of media with 100 units of IL2 per mL to        make final IL2 concentration 50 units/mL.        D22 Coating OKT3/CD28 plate        Prepared OKT3 and CD28 antibodies by adding 5 ug each of OKT3        and CD28 antibodies to 5 mL of sterile water. Added 0.5 mL of        this mixture into each well of a non-tissue culture treated 24        well plate (24-w-p). Incubated at 4° C. overnight.        D23 Activated patient 1 OKT3 blasts by thawing out a frozen vial        with 20 million cells, then plated them on OKT3/CD28 coated        plate.        Transduced patient 1 LCL by spinning down 2 million of LCL        leaving ˜50 ul of media left, then added 2 ul of Ad-LMP1-LMP2 at        5×10{circumflex over ( )}12 vp/mL for MOI of 5000. Flicked tube        every ˜15 min, and after 1.5 hrs re-suspended in 5 mL of        complete RPMI media.        D24 Culture day 16: replaced media with fresh media without any        cytokines. Incubated at 37° C. Coated plate for ELISpot assay on        D17:

Preweted a 96-w immobilon-P membrane plate with 50 ul of 35% EtOH perwell. Washed with PBS.

Made IFNy solution by adding 100 ug purified mouse anti-human IFNÅγ1-D1K antibody to 10 mL of coating buffer. Added 100 ul per well to coatplate. Store at 4° C. overnight.

D25 Culture Day 17

Harvested and counted patient 1 day 17 T cells:

Condition GMP: 8.2 million

Condition DC(px): 5.5 million

Condition Px: 15.6 million

Harvested, irradiated at 30 Gy and counted OKT3 blasts: 13.6 million

Harvested, irradiated at 40 Gy and counted patient 1 LCL: 1.2 million

Harvested, irradiated at 100 Gy and counted aK562cs: 22.5 million.

Setting up ELISpot assay to detect IFNγ release:

Blocked a 96-w-plate (with membrane coated with IFNy primary antibodyfrom the day before) with T cell media for 1 hr at 37[*]° C.

Prepared responder cells: Took out ˜1.5 million of T cells from GMPcondition, 0.8 million from DC(px) condition, and 0.8 million from Pxcondition, spun down, and re-suspended at 0.5 million per mL.

Prepared pepmix solution by adding 4 u1 of pepmix stock in DMSO (0.2ug/ul) into 800 u1 of T cell media the following antigens: EBNA1, LMP1,LMP2, and added 1.5 u1 of pepmix stock in DMSO into 300 ul of T cellmedia of the following antigens EBNA3a, EBNA3b, EBNA3c, Bzlfl, NY-ES01(irrelevant antigen for negative control), staph aureus super-antigen(positive control). Prepared patient 1 LCL by taking out ⁻1 million ofcells, spun down, resuspended in 1 million per mL. Added these antigensand targets to the responder T cells as following with each spacerepresents 2 wells duplicates:

GMP LMP2 EBNA3a con- EBNA1 LMP1 (−)ve (+)ve dition Bzlf1 LCL controlcontrol EBNA3b EBNA3c DC(px) EBNA1 LMP1 LMP2 LCL (−)ve (+)ve controlcontrol Px EBNA1 LMP1 LMP2 LCL (−)ve (+)ve control control

Added 100 ul or 50,000 cells into each well, with GMP 24 wells, DC(px)and Px each with 12 wells.

Incubated overnight at 37° C.

Optional Steps

Third stimulation:

Pulsing of OKT3 blasts with pepmixes:

Diluted EBNA1, LMP1 and LMP2 pepmixes by adding 1 ul of stock pepmixsolution (in DMSO) each into 200 ul of sterile PBS.

Spun down OKT3 blasts and aspirated all but ˜50 ul of media. Loosenedpellets by flicking the tubes. Added ˜20 ul of diluted pepmix to thepellets, incubated at 37[*]° C. for 30 min with occasional flicking.Washed with 20 mL of PBS, spun down.

Re-suspended OKT3 blasts at concentration of million per mL, thenfurther dilute out to 0.4 million per mL.

Condition GMP:

Spun down irradiated LCL and re-suspended in 10 mL of CTL media forconcentration of 0.12 million per mL. Plated out 0.1 million or 1 mLeach into 6 wells of a 24-w-p. Spun down 3 million of D17 T cells forGMP condition, re-suspended in 6 mL for and added 1 mL to each of the 6wells with LCL. T cell to LCL ratio was about 4:1.Incubated at 37° C. Froze down the rest of T cellsCondition DC(px):Spun down 2 million of DC(px) day 17 T cells, re-suspended in 10 mL of Tcell media with 2×1L4 and IL7 concentration at 20 ng/mL. Plated out 1 mLper well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3 blasts, thenadded 0.5 mL of aK562cs into each of the well to make a final volume of2 mL per well, with T cell: OKT3 blasts:aK562cs ratio of 2:2:5.Incubated at 37° C. (increased T cell and OKT3 blasts concentrationsince the growth after second stimulation was not great). Froze down therest of T cells.Condition Px:Spun down 2 million of Px day 17 T cells, re-suspended in 10 mL of Tcell media with 2×1L4 and 1L7 concentration at 20 ng/mL. Plated out 1 mLper well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3 blasts, thenadded 0.5 mL of aK562cs into each of the well to make a final volume of2 mL per well, with T cell: OKT3 blasts:aK562cs ratio of 2:2:5.Incubated at 37° C. (Increased T cell and OKT3 blasts concentration tomatch with DC(px) condition). Froze down the rest of T cells.D29 Developed ELISpot IFNγ plate.Prepared secondary IFNγ antibody (7B6-1 biotin) by adding 10 ul ofantibody to 10 mL of PBS+0.5% BSA, then filtered through a 0.2 ul filterto get rid of clumps of conjugated antibodies to prevent non-specificbinding. Washed plate 6× with 100 ul of PBS+0.05% Tween per well eachtime. Added 100 ul of this secondary antibody solution to each well.Incubated at 37° C. for 2 hours.After 1.5 hours prepared 10 mL of Avidin-Peroxidase complex solution inPBS+0.05% Tween, mixed, and incubated at room temperature. At the end ofthe 2 hr, washed plate 6× with 100 ul PBS+0.05% Tween per well eachtime. Added 100 ul Avidin-Peroxidase complex solution to each well,incubated at room temperature for 1 hour.At the end of the incubation, prepared AEC substrate by first dissolvingAEC tablet into 2.5 mL Dimethylformamide, then added 47.5 mL acetatebuffer (4.6 mL 0.1N acetic acid, 11 mL sodium acetate, 47 mL water),next added 25 ul 30% hydrogen peroxide, mixed & filtered with a 0.45 ulfilter. Washed plates with PBS+0.05% Tween, repeated 3×, washed withPBS, repeated 3×, added AEC substrate and let develop for up to 4minutes. Stopped reaction by rinsing with water. Peeled of the back anddry the membranes. Punch out results and sent for counting.D31 Added IL2 to culture day 20 by replacing 1 mL of media in each wellwith 1 mL of media with 100 units of IL2 per mL to make final IL2concentration 50 units/mL.D32 Coating OKT3/CD28 platePrepared OKT3 and CD28 antibodies by adding 5 ug each of OKT3 and CD28antibodies to 5 mL of sterile water. Added 0.5 mL of this mixture intoeach well of a non-tissue culture treated 24 well plate (24-w-p).Incubated at 4° C. overnight.D33 Activated patient 1 OKT3 blasts by plated on-going patient 1 OKT3blast culture on OKT3/CD28 coated plate at ˜1 million per well.Transduced patient 1 LCL by spinning down 2 million of LCL leaving^(˜)50 u1 of media left, then adding 2 ul of Ad-LMP1-LMP2 at 5×10″12vp/mL for MOI of 5000. Flicked tube every ˜15 min, and after 1.5 hrsre-suspended in 5 mL of complete RPMI media.D34 Culture day 23: replaced media with fresh media without anycytokines and split any wells that is over ˜4 million cells. Incubatedat 37° C.Coated plate for ELISpot assay:

Prewetted a 96-w immobilon-P membrane plate with 50 ul of 35% EtOH perwell. Washed with PBS.

Prepared IFNγ solution by adding 100 ug purified mouse anti-human IFNγ1-D1K antibody to 10 mL of coating buffer. Added 100 ul per well to coatplate. Stored at 4° C. overnight.

D35 Culture day 24

Harvested patient 1 day 24 T cell culture and counted:

Condition GMP: 22.2 million

Condition DC(px): 39.8 million

Condition Px: 16.2 million

Harvested OKT3 blasts, irradiated at 30 Gy and counted: 9.8 million

Harvested patient 1 LCL, irradiated at 40 Gy and counted: 2.3 million

Harvested aK562cs, irradiated at 100 Gy and counted: 20 million

Setting up ELISpot assay to detect IFNγ release:

Blocked a coated 96-w-plate with T cell media for 1 hr at 37° C.

Prepared responder cells: Took out ⁻2.5 million each of T cells from GMPcondition, DC(px) condition, and Px condition, spun down, andre-suspended at 1 million per mL.

Prepared pepmix solution by adding 4 ul of pepmix stock in DMSO (0.2ug/ul) into 800 ul of T cell media the following antigens: EBNA1, LMP1,LMP2, and adding 1.5 ul of pepmix stock in DMSO into 300 u1 of T cellmedia of the following antigens: EBNA3a, EBNA3b, EBNA3c, Bzlf1, NY-ESO1(irrelevant antigen for negative control), staph aureus super antigen(positive control). Prepared patient 1 LCL by taking out ˜1 million ofcells, spun down, re-suspended in 1 million per mL. Added these antigensand targets to the responder T cells as following with each spacerepresents 2 wells (duplicates):

LMP2 EBNA3a GMP EBNA1 LMP1 (−)ve (+)ve condition Bzlf1 LCL controlcontrol EBNA3b EBNA3c DC(px) EBNA1 LMP1 LMP2 LCL (−)ve (+)ve controlcontrol Px EBNA1 LMP1 LMP2 LCL (−)ve (+)ve control control

Added 100 ul or 50,000 cells into each well, with GMP 24 wells, DC(px)and Px each with 12 wells.

Incubated overnight at 37° C.

Setting up co-culture of lymphoma patient 1 T cells and autologous LCL

For each condition, added T cell and LCL to a well of a 24-w-p to theratios below:

T cell to 40:1 20:1 10:1 5:1 1:1 1:1 allo LCL ratio LCL T cell 0.5 0.50.5 0.5 0.5 0.5 count(million) LCL cell 0.0125 0.025 0.05 0.1 0.5 0.5count(million)Added IL2 to the culture for final concentration of 50 u/mL and mixedgently with transfer pipet. Phenotyped day 0 by taking out 200 ul ofeach culture, washed with 3 mL of PBS+0.5% FBS, spun down, aspiratedsupernatant and added 5 ul each of the following antibodies: CD19-PE,CD56-FITC, and CD3-PerCP. Incubated at 4° C. for 1 hour. Washed with 3mL PBS, spun down, aspirated supernatant and added 250 ul of cytofix pertube and 50 ul of CountBright beads. Analyzed with a flow cytometer.Fourth stimulation:Pulsing of OKT3 blasts with pepmixes:

Diluted EBNA1, LMP1 and LMP2 pepmixes by adding 1 ul of stock pepmixsolution (in DMSO) each into 200 ul of sterile PBS.

Spun down OKT3 blasts and aspirated all but ˜50 ul of media. Loosenedpellets by flicking the tubes. Added ˜20 ul of diluted pepmix to thepellets, incubated at 37° C. for 30 min with occasional flicking. Washedwith 20 mL of PBS, spun down.

Re-suspended OKT3 blasts at concentration of million per mL, thenfurther diluted out to 0.4 million per mL.

Re-suspended aK562cs at 2 million per mL.

Condition GMP:

Spun down irradiated LCL and re-suspended in 18.4 mL of CTL media forconcentration of 0.12 million per mL. Plated out 0.12 million or 1 mLeach into 10 wells of a 24-w-p. Spun down 5 million of D24 T cells forGMP condition, re-suspended in 10 mL for and added 1 mL to each of the10 wells with LCL. T cell to LCL ratio was about 4:1.Incubated at 37° C. Froze down the rest of T cells.Condition DC(px):Spun down 2 million of DC(px) day 24 T cells, re-suspended in 10 mL of Tcell media with 2×1L4 and IL7 concentration at 20 ng/mL. Plated out 1 mLper well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3 blasts, thenadded 0.5 mL of aK562cs at 2 million per mL into each of the well tomake a final volume of 2 mL per well, with T cell: OKT3 blasts:aK562csratio of 1:1:5. Incubated at 37° C. Froze down the rest of T cellsCondition Px:Spun down 2 million of Px day 24 T cells, re-suspended in 10 mL of Tcell media with 2× 1L4 and IL7 concentration at 20 ng/mL. Plated out 1ml per well of a 24-w-p. Added 0.5 mL of pepmix pulsed OKT3 blasts, thenadded 0.5 mL of aK562cs into each of the well to make a final volume of2 mL per well, with T cell: OKT3 blasts:aK562cs ratio of 1:1:5.Incubated at 37° C. Frozen down the rest of T cells.D36 Developed ELISpot IFNγ plate.Prepared secondary IFNγ antibody (7B6-1 biotin) by adding 10 ul ofantibody to 10 mL of PBS+0.5% BSA, then filtered through a 0.2 ul filterto get rid of clumps of conjugated antibodies to prevent non-specificbinding: Washed plate 6× with 100 ul of PBS+0.05% Tween per well eachtime. Added 100 ul of this secondary antibody solution to each well.Incubated at 37° C. for 2 hours.After 1.5 hours prepared 10 mL of Avidin-Peroxidase complex solution inPBS+0.05% Tween, mixed, and incubated at room temperature. At the end ofthe 2 hr, washed plate 6× with 100 ul PBS+0.05% Tween per well eachtime. Added 100 ul Avidin-Peroxidase complex solution to each well,incubated at room temperature for 1 hour.At the end of the incubation, prepared AEC substrate by first dissolvingAEC tablet into 2.5 mL Dimethylformamide, then added 47.5 mL acetatebuffer (4.6 mL 0.1N acetic acid, 11 mL sodium acetate, 47 mL water),next added 25 ul 30% hydrogen peroxide, mixed & filtered with a 0.45 ulfilter. Washed plates with PBS+0.05% Tween, repeated 3×, washed withPBS, repeated 3×, added AEC substrate and let develop for up to 4minutes. Stopped reaction by rinsing with water. Peeled of the back anddry the membranes. Punched out results and sent for counting. Co-cultureday 2 phenotyping

Replaced 1 mL of media with 1 mL of fresh t cell media with 100 units ofIL2.

Phenotyped day 2 by taking out 200 ul of each condition, wash with 3 mLof PBS+0.5% FBS, spun down, aspirated supernatant and added 5 ul each ofthe following antibodies: CD19-PE, CD56-FITC, and CD3-PerCP. Incubatedat 4° C. for 1 hour. Wash with 3 mL PBS, spun down, aspiratedsupernatant and added 250 ul of cytofix per tube and 50 ul ofCountBright beads. Analyzed with a flow cytometer.

D38 Co-culture day 4 phenotyping

Took out 200 u1 of each condition, washed with 3 mL of PBS+0.5% FBS,spun down, aspirated supernatant and added 10 ulj## each of thefollowing antibodies: CD19-PE, CD56-FITC, and CD3-PerCP (increasedantibody amount to match with increased cell counts), Incubated at 4° C.for 1 hour. Washed with 3 mL PBS, spun down, aspirated supernatant andadded 250 u1 of cytofix per tube and 50 u1 of CountBright beads.Analyzed with a flow cytometer.Added IL2 to culture day 20 by replacing 1 mL of media in each well with1 mL of media with 100 units of 1L2 per mL to make final IL-2concentration 50 units/mL. Spit confluent wells.D39 Co-culture day 5 phenotypingTook out 200 u1 of each condition, washed with 3 mL of PBS+0.5% FBS,spun down, aspirated supernatant and added 10 ul each of the followingantibodies: CD19-PE, CD56-FITC, and CD3-PerCP. Incubated at 4° C. for 1hour. Washed with 3 mL PBS, spun down, aspirated supernatant and added250 u1 of cytofix per tube and 50 u1 of CountBright beads. Analyzed witha flow cytometer.D41 Culture day 30: replaced media with fresh media without anycytokines and split any wells that is over ⁻4 million cells. Incubatedat 37° C.Coated plate for ELISpot assay:

Prewetede a 96-w immobilon-P membrane plate with 50 u1 of 35% EtOH perwell. Washed with PBS.

Prepared IFNy solution by adding 100 ug purified mouse anti-human IFNy1-D1K antibody to 10 mL of coating buffer. Added 100 u1 per well to coatplate. Stored at 4° C. overnight.

D42 Culture day 31

Harvested patient 1 day 31 T cell culture and counted:

Condition GMP: 17.2 million; Condition DC(px): 20.6 million; ConditionPx: 26.8 million

Results

Table 2: Whole culture expansion: Compared to current standard protocol(GMP), cultures set up with KATpx expanded just as well, if not better,by the end of the 4th stimulation.

TABLE 2 Growth in million of cells: DO D 9 D 16 D 23 D 30 Ad-DC 1 2.138.317143 61.54686 211.7212 DC 1 0.6 1.375 27.3625 281.8338 Px 1 1.013335.269333 42.6816 571.9334We have optimized a novel antigen presentation complex, KATpx, whichproduced equally good or better expansion and higher T cell antigenspecific frequencies against EBNA1, LMP1 and LMP2 in a lymphoma patient.These cells efficiently eliminate tumor cells in co-culture and thiskilling was HLA specific. Moreover, using this approach we eliminatedthe need for LCL and Adenoviral vector, thus reducing generation time aswell as activation of T cells specific for bystander antigens expressedby LCL and Adenoviral vector.It is envisaged that more than one embodiment described herein may becombined, as technically appropriate. In the context of thisspecification “comprising” is to be interpreted as “including”. Aspectsof the disclosure comprising certain elements are also intended toextend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements. All references referred to hereinare specifically incorporated by reference.

The invention claimed is:
 1. A process for in vitro expansion of antigenspecific T cells, comprising steps of: a) culturing a population ofperipheral blood mononuclear cells (PMBCs) in the presence of: i) apeptide or a peptide mix relevant to one or more target antigens ORdendritic cells which have been pulsed with the peptide or the peptidemix, and ii) at least one cytokine, thereby obtaining a population of Tcells; and b) culturing the population of T cells in the presence of:antigen presenting cells (APCs) which have been pulsed with the peptideor the peptide mix, wherein the APCs are selected from the groupconsisting of: (1) dendritic cells; and (2) antigen presenting T cellsautologous to the PMBCs; and an artificial co-stimulatory factor,characterized in that the process does not employ a live virus; a viralvector; or DNA or RNA encoding the target antigen(s).
 2. The processaccording to claim 1, further comprising repeating step b) until adesired quantity of antigen-specific T cells is obtained.
 3. The processaccording to claim 1 wherein the population of PBMCs is cultured for 12or fewer days.
 4. The process according to claim 1, wherein thepopulation of T cells is cultured for 12 or fewer days.
 5. The processaccording to claim 1, which is performed in a vessel comprising a gaspermeable culture surface.
 6. The process according to claim 1, whereinthe one or more target antigens are selected from the group consistingof antigens of an Epstein-Barr Virus, a Vaccinia Virus, and a VaricellaZoster Virus.
 7. The process according to claim 1, wherein the peptidemix comprises between 2 and 1000 peptides.
 8. The process according toclaim 1, wherein the peptides are about 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 amino acids in length.
 9. The process according to claim 1,wherein the antigen is LMP1.
 10. The process according to claim 1,wherein the antigen is LMP2.
 11. The process according to claim 1,wherein the antigen is EBNAI.
 12. The process according to claim 1,wherein the antigen is BARFI.
 13. The process according to claim 1,wherein the at least one cytokine present in step a) is IL-4.
 14. Theprocess according to claim 1, wherein the at least one cytokine presentin step a) is IL-7.
 15. The process according to claim 1, wherein stepb) further comprises at least one cytokine, wherein the at least onecytokine present in step b) is IL-15.
 16. The process according to claim1, wherein the artificial co-stimulatory factor is a cell engineered toexpress one or more co-stimulatory molecules on its surface.
 17. Theprocess according to claim 16, wherein the one or more co-stimulatorymolecules are independently selected from CD80, CD86, CD83, OX-40 ligandand 41BB-ligand or a fragment thereof.
 18. The process according toclaim 1, wherein the cell comprises CD80, CD86, CD83, OX-40 ligand and41BB-ligand.
 19. The process according to claim 1, wherein the processis performed in a GRex™ system.
 20. The process according to claim 8,wherein peptides in the peptide mix overlap by 2, 3, 45, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 or more amino acids.
 21. The process according toclaim 1, wherein the antigen specific T cells are allogeneic.